Methods for detecting a mycobacterium tuberculosis infection

ABSTRACT

Methods for detecting an infection with Mtb in a subject are disclosed. The methods include detecting the presence of CD8 +  T cells that specifically recognize an Mtb polypeptide. The methods include in vitro assays for detecting the presence of CD8 +  T cells in a biological sample, and in vivo assays that detect a delayed type hypersensitivity reaction. The methods also include detecting Mtb polypeptides and polynucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the §371 U.S. National Stage of International Application No.PCT/US2010/057503, filed Nov. 19, 2010, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 61/263,206, filed Nov. 20, 2009, which isincorporated herein by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No.HSSN266200400081C awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD

This application relates to the field of immunology, more specificallyto methods for detecting a Mycobacterium tuberculosis (Mtb) infection ina subject.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on May 15, 2012,and is 120,066 bytes, which is incorporated by reference herein.

BACKGROUND

Mycobacterium is a genus of aerobic intracellular bacterial organismsthat, upon infection of a host, survive within endosomal compartments ofmonocytes and macrophages. Human mycobacterial diseases includetuberculosis (caused by M. tuberculosis), leprosy (caused by M. leprae),Bairnsdale ulcers (caused by M. ulcerans), and various infections causedby M. marinum, M. kansasii, M. scrofulaceum, M. szulgai, M. xenopi, M.fortuitum, M. chelonae, M. haemophilum and M. intracellulare (seeWolinsky, Chapter 37 in Microbiology: Including Immunology and MolecularGenetics, 3rd Ed., Harper & Row, Philadelphia, 1980).

One third of the world's population harbors M. tuberculosis and is atrisk for developing tuberculosis (TB). In immunocompromised patients,tuberculosis is increasing at a nearly logarithmic rate, and multidrugresistant strains are appearing. In addition, mycobacterial strainswhich were previously considered to be nonpathogenic strains (e.g., M.avium) have now become major killers of immunosuppressed AIDS patients.Moreover, current mycobacterial vaccines are either inadequate (such asthe BCG vaccine for M. tuberculosis) or unavailable (such as for M.leprae) (Kaufmann, S., Microbiol. Sci. 4:324-328, 1987; U.S. Congress,Office of Technology Assessment, The Continuing Challenge ofTuberculosis, pp. 62-67, OTA-H-574, U.S. Government Printing Office,Washington, D.C., 1993).

Inhibiting the spread of tuberculosis requires effective vaccination andaccurate, early diagnosis of the disease. Currently, vaccination withlive bacteria is the most efficient method for inducing protectiveimmunity. The most common mycobacterium employed for this purpose isBacillus Calmette-Guerin (BCG), an avirulent strain of Mycobacteriumbovis. However, the safety and efficacy of BCG is a source ofcontroversy and some countries, such as the United States, do notvaccinate the general public.

Mycobacterium tuberculosis (Mtb)-specific CD4⁺ and CD8⁺ T cells arecritical for the effective control of Mtb infection. In the mouse model,passive transfer of CD4⁺ T cells to sublethally irradiated animalsrenders them less susceptible to Mtb infection (Orme, J. Immunol.140:3589-3593, 1988). Mice in which the gene(s) for CD4 (CD4^(−/−)) orfor MHC Class II molecules are disrupted, as well as wild-type micedepleted of CD4⁺ T cells, demonstrate increased susceptibility to Mtbinfection (Flory et al., J. Leukoc. Biol. 51:225-229, 1992). In humans,human immunodeficiency virus-infected individuals are exquisitelysusceptible to developing TB after exposure to Mtb, supporting anessential role for CD4⁺ T cells (Hirsch et al., J. Infect. Dis.180:2069-2073, 1999). CD8⁺ T cells are also important for effective Tcell immunity (see Lazarevic and Flynn, Am. J. Respir. Crit. Care Med.166:1116-1121, 2002). In humans, Mtb-specific CD8⁺ T cells have beenidentified in Mtb-infected individuals and include CD8⁺ T cells that areboth classically HLA-Ia restricted (see, for example, Lewinsohn et al.,J. Immunol. 165:925-930, 2000) and nonclassically restricted by theHLA-Ib molecule HLA-E (Lewinsohn et al., J. Exp. Med. 187:1633-1640,1998). However, there are no vaccines or therapeutic strategies thateffectively induce an immune response, such as a CD8 response, to Mtb.

Diagnosis of tuberculosis is commonly achieved using a skin test, whichinvolves intradermal exposure to tuberculin PPD (protein-purifiedderivative). Antigen-specific T cell responses result in measurableinduration at the injection site by 48 to 72 hours after injection,which indicates exposure to Mycobacterial antigens. However, thesensitivity and specificity of this test are not ideal; individualsvaccinated with BCG cannot be distinguished from infected individuals.Furthermore, it is very difficult to diagnose TB in children because Mtbcannot be cultured from children in the majority of cases. In bothchildren and adults, delays and missed diagnosis result in increasedmorbidity and mortality.

SUMMARY

Accordingly, there is a need in the art for improved diagnostic methodsfor detecting tuberculosis.

Methods for diagnosing an infection with Mtb are disclosed herein. Themethods include detecting CD8⁺ T cells and/or CD4⁺ T cells thatspecifically bind an Mtb polypeptide of interest. The methods alsoinclude detecting a delayed type hypersensitivity reaction in a subjectand/or include detecting specific Mtb polypeptides and polynucleotides.The disclosed assays can be used individually or in combination. The Mtbinfection can be a latent or active infection.

In several embodiments, methods are provided for detecting Mycobacteriumtuberculosis in a subject. These methods include contacting a biologicalsample from the subject comprising T cells, such as CD8⁺ T cells and/orCD4⁺ T cells, with one or more Mycobacterium polypeptides, or an antigenpresenting cell (APC) presenting the one or more Mycobacteriumpolypeptides. The one or more Mycobacterium polypeptides include anamino acid sequence set forth as (a) one of the amino acid sequences setforth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9; SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, or (b) atleast nine to twenty consecutive amino acids of at least one of theamino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQID NO: 18, wherein the nine to twenty consecutive amino acidsspecifically bind major histocompatibility complex (MHC) class I. It isdetermined whether the T cells specifically recognize the Mycobacteriumpolypeptide.

In additional embodiments, methods are provided for detectingMycobacterium tuberculosis in a subject, wherein the methods includeadministering to the subject an effective amount of a Mycobacteriumpolypeptide into the skin, subcutaneously or intradermally. TheMycobacterium polypeptide includes an amino acid sequence set forth as(a) one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQID NO: 17, or SEQ ID NO: 18; or (b) at least nine to twenty consecutiveamino acids of at least one of the amino acid sequences set forth as SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18, wherein the nine to twentyconsecutive amino acids specifically bind major histocompatibilitycomplex (MHC) class I. In some examples, the polypeptide includes aconservative variant of the polypeptide (for example, one or moreconservative amino acid substitutions). The presence of T cells thatspecifically recognize the Mycobacterium polypeptide are detected in thesubject.

In further embodiments, methods are disclosed for detecting aMycobacterium tuberculosis infection in a subject, wherein the methodsinclude detecting the presence of a Mycobacterium polypeptide or apolynucleotide encoding the polypeptide in a sample from the subject.The Mycobacterium polypeptide includes an amino acid sequence set forthas one of the amino acid sequences set forth as SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, or SEQ ID NO: 18.

Additionally, reagents for the detection of a Mycobacterium infection ina subject are described.

The foregoing and other features will become more apparent from thefollowing detailed description, which proceeds with reference to theaccompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a pair of bar graphs showing percent positive samples in theELISPOT assay for the indicated antigens in individuals with latent oractive TB.

FIG. 1B is a pair of graphs showing spot forming units (SFU) for eachantigen in the ELISPOT assay. The antigens are listed in Table 2.

FIG. 2A is a bar graph showing percent positive samples for fiveselected antigens by CD8 ELISPOT assay in individuals with latent oractive TB.

FIG. 2B is a graph showing SFU/250,000 CD4/CD56 depleted PBMC by ELISPOTassay. For each antigen, “L” indicates individuals with latent TBinfection and “A” indicates individuals with active TB infection.

FIG. 3 is a graph showing SFU for each antigen in the ELISPOT assay.

FIG. 4 is a graph showing SFU for peptides covering amino acids 137-151of Rv3136.

SEQUENCE LISTING

The nucleic and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases, and one letter code for amino acids.Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Oct. 29, 2010,and is 117 kilobytes, which is incorporated by reference herein.

SEQ ID NOs: 1-18 are the amino acid sequences of Mtb polypeptides.

SEQ ID NOs: 19-36 are the nucleic acid sequences of polynucleotidesencoding the Mtb polypeptides.

DETAILED DESCRIPTION

Methods for detecting an infection with Mtb in a subject are disclosedherein. The methods include detecting the presence of T cells, such as,but not limited to, CD8⁺ T cells, that specifically recognize a Mtbpolypeptide. The methods include in vitro assays for detecting thepresence of CD8⁺ T cells in a biological sample, and in vivo assays thatdetect a delayed type hypersensitivity reaction. The methods can alsoinclude detecting Mtb polypeptides and polynucleotides. Reagents for thedetection of an Mtb infection are also disclosed.

I. Abbreviations

APC: antigen presenting cell

BCG: Bacillus Calmette-Guerin

DC: dendritic cell

HLA: human leukocyte antigen

IFN-γ: interferon-γ

MHC: major histocompatibility complex

Mtb: Mycobacterium tuberculosis

TB: tuberculosis

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Amplification: Use of a technique that increases the number of copies ofa nucleic acid molecule (e.g., a DNA or RNA molecule) in a specimen. Anexample of amplification is the polymerase chain reaction, in which abiological sample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification canbe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: Immunoglobulin molecules and immunologically active portionsof immunoglobulin molecules, i.e., molecules that contain an antigenbinding site that specifically binds (immunoreacts with) an antigen,such as an Mtb polypeptide.

A naturally occurring antibody (e.g., IgG, IgM, IgD) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. However, it has been shown that theantigen-binding function of an antibody can be performed by fragments ofa naturally occurring antibody. Thus, these antigen-binding fragmentsare also intended to be designated by the term “antibody.” Specific,non-limiting examples of binding fragments encompassed within the termantibody include (i) a Fab fragment consisting of the V_(L), V_(H),C_(L) and C_(H1) domains; (ii) an F_(d) fragment consisting of the V_(H)and C_(H1) domains; (iii) an Fv fragment consisting of the V_(L) andV_(H) domains of a single arm of an antibody, (iv) a dAb fragment (Wardet al., Nature 341:544-546, 1989) which consists of a V_(H) domain; (v)an isolated complementarity determining region (CDR); and (vi) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region.

Immunoglobulins and certain variants thereof are known and many havebeen prepared in recombinant cell culture (e.g., see U.S. Pat. Nos.4,745,055 and 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP125,023; Falkner et al., Nature 298:286, 1982; Morrison, J. Immunol.123:793, 1979; Morrison et al., Ann. Rev. Immunol. 2:239, 1984).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. “Epitope” or “antigenicdeterminant” refers to a site on an antigen to which B and/or T cellsrespond. In one embodiment, T cells respond to the epitope, when theepitope is presented in conjunction with an MHC molecule. Epitopes canbe formed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5, about 9, or about 8-10 aminoacids in a unique spatial conformation. Methods of determining spatialconformation of epitopes include, for example, x-ray crystallography and2-dimensional nuclear magnetic resonance.

An antigen can be a tissue-specific antigen, or a disease-specificantigen. These terms are not exclusive, as a tissue-specific antigen canalso be a disease-specific antigen. A tissue-specific antigen isexpressed in a limited number of tissues, such as a single tissue. Atissue-specific antigen may be expressed by more than one tissue, suchas, but not limited to, an antigen that is expressed in more than onereproductive tissue, such as in both prostate and uterine tissue. Adisease-specific antigen is expressed coincidentally with a diseaseprocess. Specific non-limiting examples of a disease-specific antigenare an antigen whose expression correlates with, or is predictive of,tuberculosis. A disease-specific antigen can be an antigen recognized byT cells or B cells.

Antigen presenting cell (APC): A cell that can present an antigen to Tcell, such that the T cells are activated. Dendritic cells (DCs) are theprinciple APCs involved in primary immune responses. Their majorfunction is to obtain antigen in tissues, migrate to lymphoid organs andpresent the antigen in order to activate T cells.

When an appropriate maturational cue is received, DCs are signaled toundergo rapid morphological and physiological changes that facilitatethe initiation and development of immune responses. Among these are theup-regulation of molecules involved in antigen presentation; productionof pro-inflammatory cytokines, including IL-12, key to the generation ofTh1 responses; and secretion of chemokines that help to drivedifferentiation, expansion, and migration of surrounding naive Th cells.Collectively, these up-regulated molecules facilitate the ability of DCsto coordinate the activation and effector function of other surroundinglymphocytes that ultimately provide protection for the host.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

CD4: Cluster of differentiation factor 4, a T cell surface protein thatmediates interaction with the MHC Class II molecule. CD4 also serves asthe primary receptor site for HIV on T cells during HIV infection. Cellsthat express CD4 are often helper T cells.

CD8: Cluster of differentiation factor 8, a T cell surface protein thatmediates interaction with the MHC Class I molecule. Cells that expressCD8 are often cytotoxic T cells. “CD8⁺ T cell mediated immunity” is animmune response implemented by presentation of antigens to CD8⁺ T cells.

Conservative variants: A substitution of an amino acid residue foranother amino acid residue having similar biochemical properties.“Conservative” amino acid substitutions are those substitutions that donot substantially affect or decrease an activity or antigenicity of theMycobacterium polypeptide. A peptide can include one or more amino acidsubstitutions, for example 1-10 conservative substitutions, 2-5conservative substitutions, 4-9 conservative substitutions, such as 1,2, 5 or 10 conservative substitutions. Specific, non-limiting examplesof a conservative substitution include the following examples (Table 1).

TABLE 1 Exemplary conservative amino acid substitutions OriginalConservative Amino Acid Substitutions Ala Ser Arg Lys Asn Gln, His AspGlu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val LysArg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr TyrTrp; Phe Val Ile; Leu

The term conservative variation also includes the use of a substitutedamino acid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide, or that an immune response can begenerated against the substituted polypeptide that is similar to theimmune response against the unsubstituted polypeptide, such as aMycobacterium antigen. Thus, in one embodiment, non-conservativesubstitutions are those that reduce an activity or antigenicity.

Consists Essentially Of/Consists Of: With regard to a polypeptide, apolypeptide consists essentially of a specified amino acid sequence ifit does not include any additional amino acid residues. However, thepolypeptide can include additional non-peptide components, such aslabels (for example, fluorescent, radioactive, or solid particlelabels), sugars or lipids. A polypeptide that consists of a specifiedamino acid sequence does not include any additional amino acid residues,nor does it include additional non-peptide components, such as lipids,sugars or labels.

Contacting: The process of incubating one agent in the presence ofanother. Thus, when a cell is contacted with an agent, the cell isincubated with the agent for a sufficient period of time for the agentand the cell to interact.

Degenerate variant: A polynucleotide encoding an epitope of an Mtbpolypeptide that includes a sequence that is degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included in this disclosure as long as the amino acidsequence of the Mtb polypeptide encoded by the nucleotide sequence isunchanged.

Dendritic cell (DC): Dendritic cells are the principle APCs involved inprimary immune responses. DCs include plasmacytoid dendritic cells andmyeloid dendritic cells. Their major function is to obtain antigen intissues, migrate to lymphoid organs and present the antigen in order toactivate T cells. Immature DCs originate in the bone marrow and residein the periphery as immature cells.

Diagnostic: Identifying the presence or nature of a pathologiccondition, such as, but not limited to, tuberculosis. Diagnostic methodsdiffer in their sensitivity and specificity. The “sensitivity” of adiagnostic assay is the percentage of diseased individuals who testpositive (percent of true positives). The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the false positive rateis defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. “Prognostic” meanspredicting the probability of development (for example, severity) of apathologic condition, such as tuberculosis.

Displaying: The process of localizing a peptide:antigen complex, or apeptide, on the outer surface of a cell where the peptide:antigencomplex or peptide is accessible to a second cell, molecules displayedby a second cell, or soluble factors. A peptide, or a peptide:antigencomplex, is “displayed” by a cell when it is present on the outersurface of the cell and is accessible to a second cell, to moleculesdisplayed by the second cell, or to soluble factors.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, e.g., that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such as a Mycobacterium polypeptide.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (e.g., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters, are included (see e.g., Bitter etal., Meth. Enzymol. 153:516-544, 1987). For example, when cloning inbacterial systems, inducible promoters such as pL of bacteriophagelambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may beused. In one embodiment, when cloning in mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences. In one embodiment, thepromoter is a cytomegalovirus promoter.

Functionally Equivalent: Sequence alterations, such as in an epitope ofan antigen, that yield the same results as described herein. Suchsequence alterations can include, but are not limited to, conservativesubstitutions, deletions, mutations, frameshifts, and insertions.

Heterologous: Originating from separate genetic sources or species. Apolypeptide that is heterologous to an Mtb polypeptide originates from anucleic acid that does not encode the Mtb polypeptide or another Mtbpolypeptide. In one specific, non-limiting example, a polypeptidecomprising nine consecutive amino acids from an Mtb polypeptide, or atmost 20 consecutive amino acids, from the Mtb polypeptide, and aheterologous amino acid sequence includes a β-galactosidase, a maltosebinding protein, and albumin, hepatitis B surface antigen, or animmunoglobulin amino acid sequence. Generally, an antibody thatspecifically binds to a protein of interest will not specifically bindto a heterologous protein.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The cell can bemammalian, such as a human cell. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. However, such progeny are included when the term“host cell” is used.

Human Leukocyte Antigen (HLA): A genetic designation of the human majorhistocompatibility complex (MHC). Individual loci are designated byuppercase letters, as in HLA-E, and alleles are designated by numbers,as in HLA-A*0201. The three main MHC class I genes are called HLA-A,HLA-B, and HLA-C. However, there are many genes that encode β2microglobulin-associated cell surface molecules that are linked to theMHC class I genes. The expression of these genes is variable, both inthe tissue distribution and the amount expressed on cells; these geneshave been termed the MHC class IB genes.

Immune response: A response of a cell of the immune system, such as a Bcell, natural killer cell, or a T cell, to a stimulus. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In one embodiment, an immune response is aT cell response, such as a Th1, Th2, or Th3 response. In anotherembodiment, an immune response is a response of a suppressor T cell.

Immunogenic composition: A composition comprising an effective amount ofan immunogenic Mtb polypeptide or a nucleic acid encoding theimmunogenic Mtb polypeptide that induces a measurable T response againstMtb, such as a CD8⁺ T cell response, or induces a measurable B cellresponse (such as production of antibodies that specifically bind an Mtbpolypeptide). For in vitro use, the immunogenic composition can consistof the isolated nucleic acid, vector including the nucleic acid/orimmunogenic peptide. For in vivo use, the immunogenic composition willtypically comprise the nucleic acid, vector including the nucleic acid,and/or immunogenic polypeptide in pharmaceutically acceptable carriersand/or other agents. An immunogenic composition can optionally includean adjuvant, a costimulatory molecule, or a nucleic acid encoding acostimulatory molecule. An Mtb polypeptide, or nucleic acid encoding thepolypeptide, can be readily tested for its ability to induce a CD8⁺ Tcell response.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence such that the peptide will bind an MHC molecule andinduce a T cell response, such as a CD8⁺ or CD4⁺ T cell response, or a Bcell response (such as antibody production) against the antigen fromwhich the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunogenic peptides can also be identified by measuring their bindingto a specific MHC protein and by their ability to stimulate CD4 and/orCD8 when presented in the context of the MHC protein. In one example, animmunogenic “Mtb peptide” is a series of contiguous amino acid residuesfrom the Mtb protein generally between 9 and 20 amino acids in length,such as about 8 to 11 residues in length.

Generally, immunogenic Mtb polypeptides can be used to induce an immuneresponse in a subject, such as a B cell response or a T cell response.In one example, an immunogenic Mtb polypeptide, when bound to a MHCClass I molecule, activates CD8⁺ T cells, such as cytotoxic Tlymphocytes (CTLs) against Mtb. Induction of CTLs using syntheticpeptides and CTL cytotoxicity assays are known in the art (see U.S. Pat.No. 5,662,907, which is incorporated herein by reference). In oneexample, an immunogenic peptide includes an allele-specific motif orother sequence such that the peptide will bind an MHC molecule andinduce a CD8⁺ response against the antigen from which the immunogenicpeptide is derived. A CD8⁺ T cell that specifically recognizes an Mtbpolypeptide is activated, proliferates, and/or secretes cytokines inresponse to that specific polypeptide, and not to other, non-relatedpolypeptides.

Interferon gamma (IFN-γ): IFN-γ is a dimeric protein with subunits of146 amino acids. The protein is glycosylated at two sites, and the pI is8.3-8.5. IFN-γ is synthesized as a precursor protein of 166 amino acidsincluding a secretory signal sequence of 23 amino acids. Two molecularforms of the biologically active protein of 20 and 25 kDa have beendescribed. Both of them are glycosylated at position 25. The 25 kDa formis also glycosylated at position 97. The observed differences of naturalIFN-γ with respect to molecular mass and charge are due to variableglycosylation patterns. 40-60 kDa forms observed under non-denaturingconditions are dimers and tetramers of IFN-γ. The human gene has alength of approximately 6 kb. It contains four exons and maps tochromosome 12q24.1.

IFN-γ can be detected by sensitive immunoassays, such as an ELISA testthat allows detection of individual cells producing IFN-γ. Minuteamounts of IFN-γ can be detected indirectly by measuring IFN-inducedproteins such as Mx protein. The induction of the synthesis of IP-10 hasalso been used to measure IFN-γ concentrations. In addition, bioassayscan be used to detect IFN-γ, such as an assay that employs induction ofindoleamine 2,3-dioxygenase activity in 2D9 cells. The production ofIFN-γ can be used to assess T cell activation, such as activation of a Tcell by a Mycobacterium antigen.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, such as otherchromosomal and extra-chromosomal DNA and RNA, proteins, and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell, as well as chemically synthesized nucleicacids or proteins, or fragments thereof.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, enzymatic linkages, and radioactive isotopes.

Linker sequence: A linker sequence is an amino acid sequence thatcovalently links two polypeptide domains. Linker sequences can beincluded in the between the Mtb epitopes disclosed herein to providerotational freedom to the linked polypeptide domains and thereby topromote proper domain folding and presentation to the MHC. By way ofexample, in a recombinant polypeptide comprising two Mtb domains, linkersequences can be provided between them, such as a polypeptide comprisingMtb polypeptide-linker-Mtb polypeptide. Linker sequences, which aregenerally between 2 and 25 amino acids in length, are well known in theart and include, but are not limited to, the glycine(4)-serine spacer(GGGGS×3) described by Chaudhary et al., Nature 339:394-397, 1989.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mycobacteria: A genus of aerobic intracellular bacterial organisms. Uponinvasion of a host, these organisms survive within endosomalcompartments of monocytes and macrophages. Human mycobacterial diseasesinclude tuberculosis (caused by M. tuberculosis), Leprosy (caused by M.leprae), Bairnsdale ulcers (caused by M. ulcerans), and other infectionsthat can be caused by M. marinum, M. kansasii, M. scrofulaceum, M.szulgai, M. xenopi, M. fortuitum, M. haemophilum, M. chelonei, and M.intracelluare. Mycobacterium strains that were previously considered tobe nonpathogenic (such as M. avium) are also now known to be majorkillers of immunosuppressed AIDS patients.

The major response to mycobacteria involves cell mediatedhypersensitivity (DTH) reactions with T cells and macrophages playingmajor roles in the intracellular killing and walling off (or containing)of the organism (granuloma formation). A major T cell response involvesCD4⁺ lymphocytes that recognize mycobacterial heat shock proteins andimmunodominant antigens.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, the openreading frames are aligned.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a polypeptide.

Peptide Modifications: Mycobacterium polypeptides include syntheticembodiments of peptides described herein. In addition, analogues(non-peptide organic molecules), derivatives (chemically functionalizedpeptide molecules obtained starting with the disclosed peptidesequences) and variants (homologs) of these proteins can be utilized inthe methods described herein. Each polypeptide of the invention iscomprised of a sequence of amino acids, which may be either L- and/orD-amino acids, naturally occurring and otherwise.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedpeptides, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the peptide sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the peptides of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

Peptidomimetic and organomimetic embodiments are envisioned, whereby thethree-dimensional arrangement of the chemical constituents of suchpeptido- and organomimetics mimic the three-dimensional arrangement ofthe peptide backbone and component amino acid side chains, resulting insuch peptido- and organomimetics of a Mycobacterium polypeptide havingmeasurable or enhanced ability to generate an immune response. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs,” in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press, Buffalo Grove, Ill., pp. 165-174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included are mimetics prepared usingsuch techniques.

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). A “peptide” is a chain of amino acids that is lessthan 100 amino acids in length. In one embodiment, a “peptide” is aportion of a polypeptide, such as about 8-11, 9-12, or about 10, 20, 30,40, 50, or 100 contiguous amino acids of a polypeptide that is greaterthan 100 amino acids in length.

Probes and primers: Nucleic acid probes and primers may readily beprepared based on the nucleic acids provided by this invention. A probecomprises an isolated nucleic acid attached to a detectable label orreporter molecule. Typical labels include radioactive isotopes, ligands,chemiluminescent agents, and enzymes. Methods for labeling and guidancein the choice of labels appropriate for various purposes are discussed,e.g., in Sambrook et al. (1989) and Ausubel et al. (1987).

Primers are short nucleic acids, preferably DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1-3,ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, and Current Protocols in Molecular Biology, ed.Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1987(with periodic updates). PCR primer pairs can be derived from a knownsequence, for example, by using computer programs intended for thatpurpose such as Primer3 (Version 0.4.0, Whitehead Institute forBiomedical Research, Cambridge, Mass.).

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription. The promoter can be a constitutive or an induciblepromoter. A specific, non-limiting example of a promoter is the HCMV IEpromoter.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified antigenpreparation is one in which the antigen is more pure than the protein inits originating environment within a cell. A preparation of an antigenis typically purified such that the antigen represents at least 50% ofthe total protein content of the preparation. However, more highlypurified preparations may be required for certain applications. Forexample, for such applications, preparations in which the antigencomprises at least 75% or at least 90% of the total protein content maybe employed.

Recombinant: A recombinant nucleic acid or polypeptide is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two or more otherwise separated segmentsof sequence. This artificial combination is often accomplished bychemical synthesis or, more commonly, by the artificial manipulation ofisolated segments of nucleic acids, e.g., by genetic engineeringtechniques.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Variants of antigen polypeptides will possess a relatively high degreeof sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. A detailed consideration of sequence alignment methods and homologycalculations was published in 1994. The NCBI Basic Local AlignmentSearch Tool (BLAST) is available from several sources, including theNational Center for Biotechnology Information (NCBI, Bethesda, Md.) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. It can be accessedat the NCBI website. A description of how to determine sequence identityusing this program is available at the NCBI website, as are the defaultparameters.

Variants of antigenic polypeptides, such as a Mycobacteriumpolypeptides, are typically characterized by possession of at least 50%sequence identity counted over the full length alignment with the aminoacid sequence of a native antigen sequence using the NCBI Blast 2.0,gapped blastp set to default parameters. Proteins with even greatersimilarity to the reference sequences will show increasing percentageidentities when assessed by this method, such as at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 90% or at least95% sequence identity. When less than the entire sequence is beingcompared for sequence identity, variants will typically possess at least75% sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI website.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes stablyreplicated by the cell, either by incorporation of the nucleic acid intothe cellular genome, or by episomal replication. As used herein, theterm transformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Tuberculosis (TB): A disease that is generally caused by Mycobacteriumtuberculosis that usually infects the lungs. However, other “atypical”mycobacteria such as M. kansasii may produce a similar clinical andpathologic appearance of disease.

Transmission of M. tuberculosis occurs by the airborne route in confinedareas with poor ventilation. In more than 90% of cases, followinginfection with M. tuberculosis, the immune system prevents developmentof disease from M. tuberculosis, often called, active tuberculosis.However, not all of the M. tuberculosis is killed, and thus tiny, hardcapsules are formed. “Primary tuberculosis” is seen as disease thatdevelops following an initial infection, usually in children. Theinitial focus of infection is a small subpleural granuloma accompaniedby granulomatous hilar lymph node infection. Together, these make up theGhon complex. In nearly all cases, these granulomas resolve and there isno further spread of the infection. “Secondary tuberculosis” is seenmostly in adults as a reactivation of previous infection (orreinfection), particularly when health status declines. Thegranulomatous inflammation is much more florid and widespread.Typically, the upper lung lobes are most affected, and cavitation canoccur. Dissemination of tuberculosis outside of the lungs can lead tothe appearance of a number of uncommon findings with characteristicpatterns that include skeletal tuberculosis, genital tract tuberculosis,urinary tract tuberculosis, central nervous system (CNS) tuberculosis,gastrointestinal tuberculosis, adrenal tuberculosis, scrofula, andcardiac tuberculosis. “Latent” tuberculosis is an Mtb infection in anindividual that can be detected by a diagnostic assay, such as, but notlimited to a tuberculin skin test (TST) wherein the infection does notproduce symptoms in that individual. “Active” tuberculosis is asymptomatic Mtb infection in a subject.

Microscopically, the inflammation produced with TB infection isgranulomatous, with epithelioid macrophages and Langhans giant cellsalong with lymphocytes, plasma cells, maybe a few polymorphonuclearcells, fibroblasts with collagen, and characteristic caseous necrosis inthe center. The inflammatory response is mediated by a type IVhypersensitivity reaction, and skin testing is based on this reaction.In some examples, tuberculosis can be diagnosed by a skin test, an acidfast stain, an auramine stain, or a combination thereof. The most commonspecimen screened is sputum, but the histologic stains can also beperformed on tissues or other body fluids.

TB is a frequent complication of HIV infection. TB infection in subjectsinfected with a human immunodeficiency virus (HIV) can spread readilyand progress rapidly to active disease. Specific symptoms of lungdisease due to Mtb infection include chronic cough and spitting blood.Other symptoms of TB disease include fatigue, loss of appetite, weightloss, fever and drenching night sweats.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergene and other genetic elements known in the art. Vectors includeplasmid vectors, including plasmids for expression in gram negative andgram positive bacterial cell. Exemplary vectors include those forexpression in E. coli and Salmonella. Vectors also include viralvectors, such as, but not limited to, retrovirus, orthopox, avipox,fowlpox, capripox, suipox, adenovirus, herpes virus, alpha virus,baculovirus, Sindbis virus, vaccinia virus, and poliovirus vectors.Vectors also include vectors for expression in yeast cells

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

III. Mycobacterium Polypeptides

It is disclosed herein that several Mycobacterium polypeptides can beused in diagnostic assays to identify subjects infected with aMycobacterium such as Mtb. In several embodiments, the polypeptidecomprises or consists of the amino acid sequence set forth as:

VPHPWDTGDHERNWQGYFIPAMSVLRNRVGARTHAELRDAENDLVEARVIELREDPNLLGDRTDLAYLRAIHRQLFQDIYVWAGDLRTVGIEKEDESFCAPGGISRPMEHVAAEIYQLDRLRAVGEGDLAGQVAYRYDYVNYAHPFREGNGRSTREFFDLLLSERGSGLDWGKTDLEELHGACHVARANSDLTGLVAMFKGILDAEPTYDF (SEQ ID NO: 1; see also TUBERCULISTNo. Rv3641c, as available on Jun. 8, 2009, incor-porated herein by reference, known as fic).MDFALLPPEVNSARMYTGPGAGSLLAAAGGWDSLAAELATTAEAYGSVLSGLAALHWRGPAAESMAVTAAPYIGWLYTTAEKTQQTAIQARAAALAFEQAYAMTLPPPVVAANRIQLLALIATNFFGQNTAAIAATEAQYAEMWAQDAAAMYGYATASAAAALLTPFSPPRQTTNPAGLTAQAAAVSQATDPLSLLIETVTQALQALTIPSFIPEDFTFLDAIFAGYATVGVTQDVESFVAGTIGAESNLGLLNVGDENPAEVTPGDFGIGELVSATSPGGGVSASGAGGAASVGNTVLASVGRANSIGQLSVPPSWAAPSTRPVSALSPAGLTTLPGTDVAEHGMPGVPGVPVAAGRASGVLPRYGVRLTVMAHPPAAG (SEQ ID NO: 2; seealso TUBERCULIST No. Rv3136, as available on Jun.8, 2009, incorporated herein by reference, known as PPE51 or PPE).MTEPRPVFAVVISAGLSAIPMVGGPLQTVFDAIEERTRHRAETTTREICESVGGADTVLSRIDKNPELEPLLSQAIEAATRTSMEAKRRLLAQAAAAALEDDQKVEPASLIVATLSQLEPVHIHALVRLAKAAKSSPDQDEIQRREVMRAASKVEPVPVLAALIQTGVAIATTTVWHGNGTGTPAEESGHILIHDVSDFGHRLLAYLRAADAGAELLILPSGGSAPTGDHPTPHPSTSR (SEQ IDNO: 3; see also TUBERCULIST No. Rv0394c, asavailable on Jun. 8, 2009, incorporated herein by reference).MADFLTLSPEVNSARMYAGGGPGSLSAAAAAWDELAAELWLAAASFESVCSGLADRWWQGPSSRMMAAQAARHTGWLAAAATQAEGAASQAQTMALAYEAAFAATVHPALVAANRALVAWLAGSNVFGQNTPAIAAAEAIYEQMWAQDVVAMLNYHAVASAVGARLRPWQQLLHELPRRLGGEHSDSTNTELANPSSTTTRITVPGASPVHAATLLPFIGRLLAARYAELNTAIGTNWFPGTTPEVVSYPATIGVLSGSLGAVDANQSIAIGQQMLHNEILAATASGQPVTVAGLSMGSMVIDRELAYLAIDPNAPPSSALTFVELAGPERGLAQTYLPVGTTIPIAGYTVGNAPESQYNTSVVYSQYDIWADPPDRPWNLLAGANALMGAAYFHDLTAYAAPQQGIEIAAVTSSLGGTTTTYMIPSPTLPLLLPLKQIGVPDWIVGGLNNVLKPLVDAGYSQYAPTAGPYFSHGNLVW (SEQ ID NO: 4;see also TUBERCULIST No. Rv3539, as available onJun. 8, 2009, incorporated herein by reference, known as PPE63 or PPE).MTLDVPVNQGHVPPGSVACCLVGVTAVADGIAGHSLSNFGALPPEINSGRMYSGPGSGPLMAAAAAWDGLAAELSSAATGYGAAISELTNMRWWSGPASDSMVAAVLPFVGWLSTTATLAEQAAMQARAAAAAFEAAFAMTVPPPAIAANRTLLMTLVDTNWFGQNTPAIATTESQYAEMWAQDAAAMYGYASAAAPATVLTPFAPPPQTTNATGLVGHATAVAALRGQHSWAAAIPWSDIQKYWMMFLGALATAEGFIYDSGGLTLNALQFVGGMLWSTALAEAGAAEAAAGAGGAAGWSAWSQLGAGPVAASATLAAKIGPMSVPPGWSAPPATPQAQTVARSIPGIRSAAEAAETSVLLRGAPTPGRSRAAHMGRRYGRRLTVMADRPNVG(SEQ ID NO: 5; see also TUBERCULIST No. Rv1706c,as available on Jun. 8, 2009, incorporated hereinby reference, known as PPE23 or PPE).MDFGALPPEINSARMYAGAGAGPMMAAGAAWNGLAAELGTTAASYESVITRLTTESWMGPASMAMVAAAQPYLAWLTYTAEAAAHAGSQAMASAAAYEAAYAMTVPPEVVAANRALLAALVATNVLGINTPAIMATEALYAEMWAQDALAMYGYAAASGAAGMLQPLSPPSQTTNPGGLAAQSAAVGSAAATAAVNQVSVADLISSLPNAVSGLASPVTSVLDSTGLSGIIADIDALLATPFVANIINSAVNTAAWYVNAAIPTAIFLANALNSGAPVAIAEGAIEAAEGAASAAAAGLADSVTPAGLGASLGEATLVGRLSVPAAWSTAAPATTAGATALEGSGWTVAAEEAGPVTGMMPGMASAAKGTGAYAGPRYGFKPTVMPKQVVV(SEQ ID NO: 6; see also TUBERCULIST No. Rv1039c,as available on Jun. 8, 2009, incorporated hereinby reference, known as PPE15 or PPE).MAHFSVLPPEINSLRMYLGAGSAPMLQAAAAWDGLAAELGTAASFSSVTTGLTGQAWQGPASAAMAAAAAPYAGFLTTASAQAQLAAGQAKAVASVFEAAKAAIVPPAAVAANREAFLALIRSNWLGLNAPWIAAVESLYEEYWAADVAAMTGYHAGASQAAAQLPLPAGLQQFLNTLPNLGIGNQGNANLGGGNTGSGNIGNGNKGSSNLGGGNIGNNNIGSGNRGSDNFGAGNVGTGNIGFGNQGPIDVNLLATPGQNNVGLGNIGNNNMGFGNTGDANTGGGNTGNGNIGGGNTGNNNFGFGNTGNNNIGIGLTGNNQMGINLAGLLNSGSGNIGIGNSGTNNIGLFNSGSGNIGVFNTGANTLVPGDLNNLGVGNSGNANIGFGNAGVLNTGFGNASILNTGLGNAGELNTGFGNAGFVNTGFDNSGNVNTGNGNSGNINTGSWNAGNVNTGFGIITDSGLTNSGFGNTGTDVSGFFNTPTGPLAVDVSGFFNTASGGTVINGQTSGIGNIGVPGTLFGSVRSGLNTGLFNMGTAISGLFNLRQLLG (SEQ ID NO: 7; see also TUBERCULIST No.Rv3558, as available on Jun. 8, 2009, incorpo-rated herein by reference, known as PPE64 or PPE).MEYLIAAQDVLVAAAADLEGIGSALAAANRAAEAPTTGLLAAGADEVSAAIASLFSGNAQAYQALSAQAAAFHQQFVRALSSAAGSYAAAEAANASPMQAVLDVVNGPTQLLLGRPLIGDGANGGPGQNGGDGGLLYGNGGNGGSSSTPGQPGGRGGAAGLIGNGGAGGAGGPGANGGAGGNGGWLYGNGGLGGNGGAATQIGGNGGNGGHGGNAGLWGNGGAGGAGAAGAAGANGQNPVSHQVTHATDGADGTTGPDGNGTDAGSGSNAVNPGVGGGAGGIGGDGTNLGQTDVSGGAGGDGGDGANFASGGAGGNGGAAQSGFGDAVGGNGGAGGNGGAGGGGGLGGAGGSANVANAGNSIGGNGGAGGNGGIGAPGGAGGAGGNANQDNPPGGNSTGGNGGAGGDGGVGASADVGGAGGFGGSGGRGGLLLGTGGAGGDGGVGGDGGIGAQGGSGGNGGNGGIGADGMANQDGDGGDGGNGGDGGAGGAGGVGGNGGTGGAGGLFGQSGSPGSGAAGGLGGAGGNGGAGGGGGTGFNPGAPGDPGTQGATGANGQHGLN (SEQ ID NO: 8; seealso TUBERCULIST No. Rv1243c, as available on Oct.6, 2009; incorporated herein by reference, known as PE_PGRS23).MVMSLMVAPELVAAAAADLTGIGQAISAANAAAAGPTTQVLAAAGDEVSAAIAALFGTHAQEYQALSARVATFHEQFVRSLTAAGSAYATAEAANASPLQALEQQVLGAINAPTQLWLGRPLIGDGVHGAPGTGQPGGAGGLLWGNGGNGGSGAAGQVGGPGGAAGLFGNGGSGGSGGAGAAGGVGGSGGWLNGNGGAGGAGGTGANGGAGGNAWLFGAGGSGGAGTNGGVGGSGGFVYGNGGAGGIGGIGGIGGNGGDAGLFGNGGAGGAGAAGLPGAAGLNGGDGSDGGNGGTGGNGGRGGLLVGNGGAGGAGGVGGDGGKGGAGDPSFAVNNGAGGNGGHGGNPGVGGAGGAGGLLAGAHGAAGATPTSGGNGGDGGIGATANSPLQAGGAGGNGGHGGLVGNGGTGGAGGAGHAGSTGATGTALQPTGGNGTNGGAGGHGGNGGNGGAQHGDGGVGGKGGAGGSGGAGGNGFDAATLGSPGADGGMGGNGGKGGDGGKAGDGGAGAAGDVTLAVNQGAGGDGGNGGEVGVGGKGGAGGVSANPALNGSAGANGTAPTSGGNGGNGGAGATPTVAGENGGAGGNGGHGGSVGNGGAGGAGGNGVAGTGLALNGGNGGNGGIGGNGGSAAGTGGDGGKGGNGGAGANGQDFSASANGANGGQGGNGGNGGIGGKGGDAFATFAKAGNGGAGGNGGNVGVAGQGGAGGKGAIPAMKGATGADGTAPTSGGDGGNGGNGASPTVAGGNGGDGGKGGSGGNVGNGGNGGAGGNGAAGQAGTPGPTSGDSGTSGTDGGAGGNGGAGGAGGTLAGHGGNGGKGGNGGQGGIGGAGERGADGAGPNANGANGENGGSGGNGGDGGAGGNGGAGGKAQAAGYTDGATGTGGDGGNGGDGGKAGDGGAGENGLNSGAMLPGGGTVGNPGTGGNGGNGGNAGVGGTGGKAGTGSLTGLDGTDGITPNGGNGGNGGNGGKGGTAGNGSGAAGGNGGNGGSGLNGGDAGNGGNGGGALNQAGFFGTGGKGGNGGNGGAGMINGGLGGFGGAGGGGAVDVAATTGGAGGNGGAGGFASTGLGGPGGAGGPGGAGDFASGVGGVGGAGGDGGAGGVGGFGGQGGIGGEGRTGGNGGSGGDGGGGISLGGNGGLGGNGGVSETGFGGAGGNGGYGGPGGPEGNGGLGGNGGAGGNGGVSTTGGDGGAGGKGGNGGDGGNVGLGGDAGSGGAGGNGGIGTDAGGAGGAGGAGGNGGSSKSTTTGNAGSGGAGGNGGTGLNGAGGAGGAGGNAGVAGVSFGNAVGGDGGNGGNGGHGGDGTTGGAGGKGGNGSSGAASGSGVVNVTAGHGGNGGNGGNGGNGSAGAGGQGGAGGSAGNGGHGGGATGGDGGNGGNGGNSGNSTGVAGLAGGAAGAGGNGGGTSSAAGHGGSGGSGGSGTTGGAGAAGGNGGAGAGGGSLSTGQSGGPRRQRWCRWQRRRWLGRQRRRRWCRWQRRCRRQRWRWRCRQRRLRRQWRQGRRRCRPWLHRRRGRQGRRWRQRRFQQRQRSRWQRR (SEQID NO: 9; see also TUBERCULIST No. Rv3345c, asavailable on Oct. 6, 2009; incorporated hereinby reference, known as PE_PGRS50).VIQTCEVELRWRASQLTLAIATCAGVALAAAVVAGRWQLIAFAAPLLGVLCSISWQRPVPVIQVHGDPDSQRCFENEHVRVTVWVTTESVDAAVELTVSALAGMQFEALESVSRRTTTVSAVAQRWGRYPIRARVAVVARGGLLMGAGTVDAAEIVVFPLTPPQSTPLPQTELLDRLGAHLTRHVGPGVEYADIRPYVPGDQLRAVNWVVSARRGRLHVTRRLTDRAADVVVLIDMYRQPAGPATEATERVVRGAAQVVQTALRNGDRAGIVALGGNRPRWLGADIGQRQFYRVLDTVLGAGEGFENTTGTLAPRAAVPAGAVVIAFSTLLDTEFALALIDLRKRGHVVVAVDVLDSCPLQDQLDPLVVRMVVALQRSAMYRDMATIGVDVLSWPADHSLQQSMGALPNRRRRGRGRASRARLP (SEQ ID NO: 10; see alsoTUBERCULIST No. Rv3163c, as available on Oct. 6,2009; incorporated herein by reference).VNRRILTLMVALVPIVVFGVLLAVVTVPFVALGPGPTFDTLGEIDGKQVVQIVGTQTYPTSGHLNMTTVSQRDGLTLGEALALWLSGQEQLMPRDLVYPPGKSREEIENDNAADFKRSEAAAEYAALGYLKYPKAVTVASVMDPGPSVDKLQAGDAIDAVDGTPVGNLDQFTALLKNTKPGQEVTIDFRRKNEPPGIAQITLGKNKDRDQGVLGIEVVDAPWAPFAVDFHLANVGGPSAGLMFSLAVVDKLTSGHLVGSTFVAGTGTIAVDGKVGQIGGITHKMAAARAAGATVFLVPAKNCYEASSDSPPGLKLVKVETLSQAVDALHAMTSGSPTPSC (SEQ IDNO: 11; see also TUBERCULIST No. Rv3194c, asavailable on Oct. 6, 2009; incorporated herein by reference).MSFVVTAPPVLASAASDLGGIASMISEANAMAAVRTTALAPAAADEVSAAIAALFSSYARDYQTLSVQVTAFHVQFAQTLTNAGQLYAVVDVGNGVLLKTEQQVLGVINAPTQTLVGRPLIGDGTHGAPGTGQNGGAGGILWGNGGNGGSGAPGQPGGRGGDAGLFGHGGHGGVGGPGIAGAAGTAGLPGGNGANGGSGGIGGAGGAGGNGGLLFGNGGAGGQGGSGGLGGSGGTGGAGMAAGPAGGTGGIGGIGGIGGAGGVGGHGSALFGHGGINGDGGTGGMGGQGGAGGNGWAAEGITVGIGEQGGQGGDGGAGGAGGIGGSAGGIGGSQGAGGHGGDGGQGGAGGSGGVGGGGAGAGGDGGAGGIGGTGGNGSIGGAAGNGGNGGRGGAGGMATAGSDGGNGGGGGNGGVGVGSAGGAGGTGGDGGAAGAGGAPGHGYFQQPAPQGLPIGTGGTGGEGGAGGAGGDGGQGDIGFDGGRGGDGGPGGGGGAGGDGSGTFNAQANNGGDGGAGGVGGAGGTGGTGGVGADGGRGGDSGRGGDGGNAGHGGAAQFSGRGAYGGEGGSGGAGGNAGGAGTGGTAGSGGAGGFGGNGADGGNGGNGGNGGFGGINGTFGTNGAGGTGGLGTLLGGHNGNIGLNGATGGIGSTTLTNATVPLQLVNTTEPVVFISLNGGQMVPVLLDTGSTGLVMDSQFLTQNFGPVIGTGTAGYAGGLTYNYNTYSTTVDFGNGLLTLPTSVNVVTSSSPGTLGNFLSRSGAVGVLGIGPNNGFPGTSSIVTAMPGLLNNGVLIDESAGILQFGPNTLTGGITISGAPISTVAVQIDNGPLQQAPVMFDSGGINGTIPSALASLPSGGFVPAGTTISVYTSDGQTLLYSYTTTATNTPFVTSGGVMNTGHVPFAQQPIYVSYSPTAIGTTTFN (SEQ IDNO: 12; see also TUBERCULIST No. Rv0977, as avail-able on Oct. 6, 2009; incorporated herein by reference).MTHDHAHSRGVPAMIKEIFAPHSHDAADSVDDTLESTAAGIRTVKISLLVLGLTALIQIVIVVMSGSVALAADTIHNFADALTAVPLWIAFALGAKPATRRYTYGFGRVEDLAGSFVVAMITMSAIIAGYEAIARLIHPQQIEHVGWVALAGLVGFIGNEWVALYRIRVGHRIGSAALIADGLHARTDGFTSLAVLCSAGGVALGFPLADPIVGLLITAAILAVLRTAARDVFRRLLDGVDPAMVDAAEQALAARPGVQAVRSVRMRWIGHRLHADAELDVDPALDLAQAHRIAHDAEHELTHTVPKLTTALIHAYPAEHGSSIPDRGRTVE (SEQ ID NO: 13;see also TUBERCULIST No. Rv2025c, as available onOct. 6, 2009; incorporated herein by reference).VVNFSVLPPEINSGRMFFGAGSGPMLAAAAAWDGLAAELGLAAESFGLVTSGLAGGSGQAWQGAAAAAMVVAAAPYAGWLAAAAARAGGAAVQAKAVAGAFEAARAAMVDPVVVAANRSAFVQLVLSNVFGQNAPAIAAAEATYEQMWAADVAAMVGYHGGASAAAAALAPWQQAVPGLSGLLGGAANAPAAAAQGAAQGLAELTLNLGVGNIGSLNLGSGNIGGTNVGSGNVGGTNLGSGNYGSLNWGSGNTGTGNAGSGNTGDYNPGSGNFGSGNFGSGNIGSLNVGSGNFGTLNLANGNNGDVNFGGGNTGDFNFGGGNNGTLNFGFGNTGSGNFGFGNTGNNNIGIGLTGDGQIGIGGLNSGTGNIGFGNSGNNNIGFFNSGDGNIGFFNSGDGNTGFGNAGNINTGFWNAGNLNTGFGSAGNGNVGIFDGGNSNSGSFNVGFQNTGFGNSGAGNTGFFNAGDSNTGFANAGNVNTGFFNGGDINTGGFNGGNVNTGFGSALTQAGANSGFGNLGTGNSGWGNSDPSGTGNSGFFNTGNGNSGFSNAGPAMLPGFNSGFANIGSFNAGIANSGNNLAGISNSGDDSSGAVNSGSQNSGAFNAGVGLSGFFR (SEQ ID NO: 14; see alsoTUBERCULIST No. Rv2356c, as available on Oct. 6,2009; incorporated herein by reference, known as PPE40).MNYSVLPPEINSLRMFTGAGSAPMLAASVAWDRLAAELAVAASSFGSVTSGLAGQSWQGAAAAAMAAAAAPYAGWLAAAAARAAGASAQAKAVASAFEAARAATVHPMLVAANRNAFVQLVLSNLFGQNAPAIAAAEAMYEQMWAADVAAMVGYHGGASAAAAQLSSWSIGLQQALPAAPSALAAAIGLGNIGVGNLGGGNTGDYNLGSGNSGNANVGSGNSGNANVGSGNDGATNLGSGNIGNTNLGSGNVGNVNLGSGNRGFGNLGNGNFGSGNLGSGNTGSTNFGGGNLGSFNLGSGNIGSSNIGFGNNGDNNLGLGNNGNNNIGFGLTGDNLVGIGALNSGIGNLGFGNSGNNNIGFFNSGNNNVGFFNSGNNNFGFGNAGDINTGFGNAGDTNTGFGNAGFFNMGIGNAGNEDMGVGNGGSFNVGVGNAGNQSVGFGNAGTLNVGFANAGSINTGFANSGSINTGGFDSGDRNTGFGSSVDQSVSSSGFGNTGMNSSGFFNTGNVSAGYGNNGDVQSGINNTNSGGFNVGFYNSGAGTVGIANSGLQTTGIANSGTLNTGVANTGDHSSGGFNQGSDQSGFFGQP(SEQ ID NO: 15; see also TUBERCULIST No. Rv3159c,as available on Oct. 6, 2009; incorporated hereinby reference, known as PPE53).MSFVFAAPEALAAAAADMAGIGSTLNAANVVAAVPTTGVLAAAADEVSTQVAALLSAHAQGYQQLSRQMMTAFHDQFVQALRASADAYATAEASAAQTMVNAVNAPARALLGHPLISADASTGGGSNALSRVQSMFLGTGGSSALGGSAAANAAASGALQLQPTGGASGLSAVGALLPRAGAAAAAALPALAAESIGNAIKNLYNAVEPWVQYGFNLTAWAVGWLPYIGILAPQINFFYYLGEPIVQAVLFNAIDFVDGTVTFSQALTNIETATAASINQFINTEINVVIRGFLPPLPPISPPGFPSLP (SEQ ID NO: 16; see also TUBERCULISTNo. Rv1172c, as available on Oct. 6, 2009;incorporated herein by reference, known as PE12).MDYAFLPPEINSARMYSGPGPNSMLVAAASWDALAAELASAAENYGSVIARLTGMHWVVGPASTSMLAMSAPYVEWLERTAAQTKQTATQARAAAAAFEQAHAMTVPPALVTGIRGAIVVETASASNTAGTPP (SEQ ID NO: 17;see also TUBERCULIST No. Rv3135, as available onJun. 8, 2009, incorporated herein by reference, known as PPE50 or PPE).LSASVSATTAHHGLPAHEVVLLLESDPYHGLSDGEAAQRLERFGPNTLAVVTRASLLARILRQFHHPLIYVLLVAGTITAGLKEFVDAAVIFGVVVINAIVGFIQESKAEAALQGLRSMVHTHAKVVREGHEHTMPSEELVPGDLVLLAAGDKVPADLRLVRQTGLSVNESALTGESTPVHKDEVALPEGTPVADRRNIAYSGTLVTAGHGAGIVVATGAETELGEIHRLVGAAEVVATPLTAKLAWFSKFLTIAILGLAALTFGVGLLRRQDAVETFTAAIALAVGAIPEGLPTAVTITLAIGMARMAKRRAVIRRLPAVETLGSTTVICADKTGTLTENQMTVQSIWTPHGEIRATGTGYAPDVLLCDTDDAPVPVNANAALRWSLLAGACSNDAALVRDGTRWQIVGDPTEGAMLVVAAKAGFNPERLATTLPQVAAIPFSSERQYMATLHRDGTDHVVLAKGAVERMLDLCGTEMGADGALRPLDRATVLRATEMLTSRGLRVLATGMGAGAGTPDDFDENVIPGSLALTGLQAMSDPPRAAAASAVAACHSAGIAVKMITGDHAGTATAIATEVGLLDNTEPAAGSVLTGAELAALSADQYPEAVDTASVFARVSPEQKLRLVQALQARGHVVAMTGDGVNDAPALRQANIGVAMGRGGTEVAKDAADMVLTDDDFATIEAAVEEGRGVFDNLTKFITWTLPTNLGEGLVILAAIAVGVALPILPTQILWINMTTAIALGLMLAFEPKEAGIMTRPPRDPDQPLLTGWLVRRTLLVSTLLVASAWWLFAWELDNGAGLHEARTAALNLFVVVEAFYLFSCRSLTRSAWRLGMFANRWIILGVSAQAIAQFAITYLPAMNMVFDTAPIDIGVWVRIFAVATAITIVVATDTLLPRIRAQPP (SEQ ID NO: 18; see also TUBERCULIST No.Rv1997, as available on Jun. 8, 2009,incorporated herein by reference, known as ctpF).

In a second embodiment, an Mtb polypeptide of use in the methodsdisclosed herein has a sequence at least 75%, 85%, 90%, 95%, 96%, 97%,98% or 99% homologous to the amino acid sequence set forth in one of SEQID NOs: 1-18. For example, the polypeptide can have an amino acidsequence at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to oneof the amino acid sequences set forth in SEQ ID NOs: 1-18. Exemplarysequences can be obtained using computer programs that are readilyavailable on the internet and the amino acid sequences set forth herein.In one example, the polypeptide retains a function of the Mtb protein,such as binding to an antibody that specifically binds the Mtb epitope.

Minor modifications of an Mtb polypeptide primary amino acid sequencesmay result in peptides which have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polypeptides produced by thesemodifications are included herein. Thus, a specific, non-limitingexample of an Mtb polypeptide is a conservative variant of the Mtbpolypeptide (such as a single conservative amino acid substitution, forexample, one or more conservative amino acid substitutions, for example1-10 conservative substitutions, 2-5 conservative substitutions, 4-9conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions). A table of conservative substitutions is providedherein. Substitutions of the amino acids sequence shown in SEQ ID NOs:1-18 can be made based on this table.

Mtb polypeptides are disclosed herein that can be used to induce animmune response to Mtb. These peptides include or consist of at leastnine amino acids, such as nine to twenty amino acids consecutive aminoacids of an Mtb polypeptide set forth above. In particular non-limitingexamples the Mtb polypeptide includes or consists of MDFALLPPEVNSARM(amino acids 1-15 of SEQ ID NO: 2) or AEMWAQDAA (amino acids 141-149 ofSEQ ID NO: 2). Specific, non-limiting examples are fifteen, fourteen,thirteen, twelve, eleven, ten, or nine consecutive amino acids of one ofthe Mtb polypeptides set forth above. In these examples, the Mtbpolypeptide does not include the full-length amino acid sequences setforth as SEQ ID NOs: 1-18.

In several embodiments, the isolated Mtb polypeptide is included in afusion protein. Thus, the fusion protein can include the Mtb polypeptide(see above) and a second heterologous moiety, such as a myc protein, anenzyme or a carrier (such as a hepatitis carrier protein or bovine serumalbumin) covalently linked to the Mtb polypeptide. In several examples,a polypeptide consisting of nine to twelve amino acids of one of theamino acid sequences set forth as SEQ ID NOs: 1-18 that bind MHC class Iis covalently linked to a carrier. In an additional example, apolypeptide consisting of one of the amino acid sequences set forth asone of SEQ ID NOs: 1-18 is covalently linked to a carrier.

In additional examples, the polypeptide can be a fusion protein and canalso include heterologous sequences to Mtb. Thus, in several specificnon-limiting examples, the immunogenic peptide is a fusion polypeptide,for example the polypeptide includes six sequential histidine residues,a β-galactosidase amino acid sequence, or an immunoglobulin amino acidsequence. The polypeptide can also be covalently linked to a carrier. Inadditional embodiments, the protein consists of the Mtb polypeptide.

The polypeptide can optionally include repetitions of one or more of theMtb polypeptides disclosed herein. In one specific, non-limitingexample, the polypeptide includes two, three, four, five, or up to tenrepetitions of one of the Mtb polypeptides described above.Alternatively, more than one polypeptide can be included in a fusionpolypeptide. Thus, in several examples, the polypeptide can include atleast two, at least three, at least four, at least five or at least sixof the amino acid sequences set forth as SEQ ID NOs: 1-18 or nine totwenty amino acids of one of the amino acid sequences set forth as SEQID NOs: 1-18 and repetitions of these sequences. A linker sequence canoptionally be included between the Mtb polypeptides.

The Mtb polypeptides disclosed herein can be chemically synthesized bystandard methods, or can be produced recombinantly. An exemplary processfor polypeptide production is described in Lu et al., FEBS Lett.429:31-35, 1998. They can also be isolated by methods includingpreparative chromatography and immunological separations. Polypeptidescan also be produced using molecular genetic techniques, such as byinserting a nucleic acid encoding Mtb or an epitope thereof into anexpression vector, introducing the expression vector into a host cell,and isolating the polypeptide (see below).

In particular embodiments provided herein, one or more of the disclosedMtb polypeptides (or fragments thereof) can be conjugated to a substrateor solid support, such as a plate or array. In one example, the plate orarray includes, consists essentially of, or consists of one (such as 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) of SEQ IDNOs: 1-18 or fragments thereof. In some examples, the plate or arrayalso includes one or more control polypeptides. Methods for selecting anappropriate substrate and constructing a plate or array are well knownto one of skill in the art (see, e.g., U.S. Pat. Nos. 5,143,854;5,405,783; 5,445,934; and 5,744,305; all incorporated herein byreference).

Polynucleotides encoding the Mtb polypeptides disclosed herein are alsoprovided. Exemplary nucleic acid sequences are set forth as SEQ ID NOs:19-36. These polynucleotides include DNA, cDNA and RNA sequences whichencode the polypeptide of interest. Silent mutations in the codingsequence result from the degeneracy (i.e., redundancy) of the geneticcode, whereby more than one codon can encode the same amino acidresidue. Tables showing the standard genetic code can be found invarious sources (e.g., L. Stryer, 1988, Biochemistry, 3^(rd) Edition, W.H. Freeman and Co., NY).

A nucleic acid encoding an Mtb polypeptide can be cloned or amplified byin vitro methods, such as the polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR) andthe Qβ replicase amplification system (QB). For example, apolynucleotide encoding the protein can be isolated by polymerase chainreaction of cDNA using primers based on the DNA sequence of themolecule. A wide variety of cloning and in vitro amplificationmethodologies are well known to persons skilled in the art. PCR methodsare described in, for example, U.S. Pat. No. 4,683,195; Mullis et al.,Cold Spring Harbor Symp. Quant. Biol. 51:263, 1987; and Erlich, ed., PCRTechnology (Stockton Press, N.Y., 1989). Polynucleotides also can beisolated by screening genomic or cDNA libraries with probes selectedfrom the sequences of the desired polynucleotide under stringenthybridization conditions.

The polynucleotides encoding an Mtb polypeptide include a recombinantDNA which is incorporated into a vector into an autonomously replicatingplasmid or virus or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (such as a cDNA) independent ofother sequences. The nucleotides of the invention can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

In one embodiment, vectors are used for expression in yeast such as S.cerevisiae or Kluyveromyces lactis. Several promoters are known to be ofuse in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive METS and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (such as AMP) forpropagation in bacteria. Plasmids for expression on K. lactis are known,such as pKLAC1. Thus, in one example, after amplification in bacteria,plasmids can be introduced into the corresponding yeast auxotrophs bymethods similar to bacterial transformation.

The Mtb polypeptides can be expressed in a variety of yeast strains. Forexample, seven pleiotropic drug-resistant transporters, YOR1, SNQ2,PDR5, YCF1, PDR10, PDR11, and PDR15, together with their activatingtranscription factors, PDR1 and PDR3, have been simultaneously deletedin yeast host cells, rendering the resultant strain sensitive to drugs.Yeast strains with altered lipid composition of the plasma membrane,such as the erg6 mutant defective in ergosterol biosynthesis, can alsobe utilized. Proteins that are highly sensitive to proteolysis can beexpressed in a yeast strain lacking the master vacuolar endopeptidasePep4, which controls the activation of other vacuolar hydrolases.Heterologous expression in strains carrying temperature-sensitive (ts)alleles of genes can be employed if the corresponding null mutant isinviable.

Viral vectors encoding the Mtb polypeptides disclosed herein can also beprepared. A number of viral vectors have been constructed, includingpolyoma, SV40 (Madzak et al., 1992, J. Gen. Virol. 73:15331536),adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol. 158:39-6;Berliner et al., 1988, BioTechniques 6:616-629; Gorziglia et al., 1992,J. Virol. 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci. USA89:2581-2584; Rosenfeld et al., 1992, Cell 68:143-155; Wilkinson et al.,1992, Nucl. Acids Res. 20:2233-2239; Stratford-Perricaudet et al., 1990,Hum. Gene Ther. 1:241-256), vaccinia virus (Mackett et al., 1992,Biotechnology 24:495-499), adeno-associated virus (Muzyczka, 1992, Curr.Top. Microbiol. Immunol. 158:91-123; On et al., 1990, Gene 89:279-282),herpes viruses including HSV and EBV (Margolskee, 1992, Curr. Top.Microbiol. Immunol. 158:67-90; Johnson et al., 1992, J. Virol.66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19; Breakfield etal., 1987, Mol. Neurobiol. 1:337-371; Fresse et al., 1990, Biochem.Pharmacol. 40:2189-2199), Sindbis viruses (Herweijer et al., 1995, Hum.Gene Ther. 6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879),alphaviruses (Schlesinger, 1993, Trends Biotechnol. 11:18-22; Frolov etal., 1996, Proc. Natl. Acad. Sci. USA 93:11371-11377) and retrovirusesof avian (Brandyopadhyay et al., 1984, Mol. Cell Biol. 4:749-754;Petropouplos et al., 1992, J. Virol. 66:3391-3397), murine (Miller,1992, Curr. Top. Microbiol. Immunol. 158:1-24; Miller et al., 1985, Mol.Cell Biol. 5:431-437; Sorge et al., 1984, Mol. Cell Biol. 4:1730-1737;Mann et al., 1985, J. Virol. 54:401-407), and human origin (Page et al.,1990, J. Virol. 64:5370-5276; Buchschalcher et al., 1992, J. Virol.66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.).

Thus, in one embodiment, the polynucleotide encoding an Mtb polypeptideis included in a viral vector. Suitable vectors include retrovirusvectors, orthopox vectors, avipox vectors, fowlpox vectors, capripoxvectors, suipox vectors, adenoviral vectors, herpes virus vectors, alphavirus vectors, baculovirus vectors, Sindbis virus vectors, vacciniavirus vectors and poliovirus vectors. Specific exemplary vectors arepoxvirus vectors such as vaccinia virus, fowlpox virus and a highlyattenuated vaccinia virus (MVA), adenovirus, baculovirus and the like.

Pox viruses useful in practicing the present methods include orthopox,suipox, avipox, and capripox virus. Orthopox include vaccinia,ectromelia, and raccoon pox. One example of an orthopox of use isvaccinia. Avipox includes fowlpox, canary pox and pigeon pox. Capripoxinclude goatpox and sheep pox. In one example, the suipox is swinepox.Examples of pox viral vectors for expression as described for example,in U.S. Pat. No. 6,165,460, which is incorporated herein by reference.Other viral vectors that can be used include other DNA viruses such asherpes virus and adenoviruses, and RNA viruses such as retroviruses andpoliovirus.

The vaccinia virus genome is known in the art. It is composed of a HINDF13L region, TK region, and an HA region. Recombinant vaccinia virus hasbeen used to incorporate an exogenous gene for expression of theexogenous gene product (see, for example, Perkus et al. Science229:981-984, 1985; Kaufman et al. Int. J. Cancer 48:900-907, 1991; Moss,Science 252:1662, 1991). A gene encoding an antigen of interest, such asan immunogenic Mtb polypeptide, can be incorporated into the HIND F13Lregion or alternatively incorporated into the TK region of recombinantvaccinia virus vector (or other nonessential regions of the vacciniavirus genome). Baxby and Paoletti (Vaccine 10:8-9, 1992) disclose theconstruction and use as a vector, of the non-replicating poxvirus,including canarypox virus, fowlpox virus and other avian species. Sutterand Moss (Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851, 1992) and Sutteret al. (Vaccine 12:1032-1040, 1994) disclose the construction and use asa vector of the non-replicating recombinant Ankara virus (MVA, modifiedvaccinia Ankara).

Suitable vectors are disclosed, for example, in U.S. Pat. No. 6,998,252,which is incorporated herein by reference. In one example, a recombinantpoxvirus, such as a recombinant vaccinia virus is synthetically modifiedby insertion of a chimeric gene containing vaccinia regulatory sequencesor DNA sequences functionally equivalent thereto flanking DNA sequenceswhich in nature are not contiguous with the flanking vaccinia regulatoryDNA sequences that encode a Mtb polypeptide. The recombinant viruscontaining such a chimeric gene is effective at expressing the Mtbpolypeptide. In one example, the vaccine viral vector comprises (A) asegment comprised of (i) a first DNA sequence encoding a Mtb polypeptideand (ii) a poxvirus promoter, wherein the poxvirus promoter is adjacentto and exerts transcriptional control over the DNA sequence encoding anMtb polypeptide; and, flanking said segment, (B) DNA from a nonessentialregion of a poxvirus genome. The viral vector can encode a selectablemarker. In one example, the poxvirus includes, for example, a thymidinekinase gene (see U.S. Pat. No. 6,998,252, which is incorporated hereinby reference).

Viral vectors, such as poxviral vectors, that encode an Mtb polypeptideinclude at least one expression control element operationally linked tothe nucleic acid sequence encoding the Mtb polypeptide. The expressioncontrol elements are inserted in the viral vector to control andregulate the expression of the nucleic acid sequence. Examples ofexpression control elements of use in these vectors includes, but is notlimited to, lac system, operator and promoter regions of phage lambda,yeast promoters and promoters derived from polyoma, adenovirus,retrovirus or SV40. Additional operational elements include, but are notlimited to, leader sequence, termination codons, polyadenylation signalsand any other sequences necessary for the appropriate transcription andsubsequent translation of the nucleic acid sequence encoding the Mtbpolypeptide in the host system. The expression vector can containadditional elements necessary for the transfer and subsequentreplication of the expression vector containing the nucleic acidsequence in the host system. Examples of such elements include, but arenot limited to, origins of replication and selectable markers. It willfurther be understood by one skilled in the art that such vectors areeasily constructed using conventional methods (Ausubel et al., (1987) in“Current Protocols in Molecular Biology,” John Wiley and Sons, New York,N.Y.) and are commercially available.

Basic techniques for preparing recombinant DNA viruses containing aheterologous DNA sequence encoding the one or more Mtb polypeptides areknown in the art. Such techniques involve, for example, homologousrecombination between the viral DNA sequences flanking the DNA sequencein a donor plasmid and homologous sequences present in the parentalvirus (Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79:7415-7419).In particular, recombinant viral vectors such as a poxviral vector canbe used in delivering the gene. The vector can be constructed forexample by steps known in the art, such as steps analogous to themethods for creating synthetic recombinants of the fowlpox virusdescribed in U.S. Pat. No. 5,093,258, incorporated herein by reference.Other techniques include using a unique restriction endonuclease sitethat is naturally present or artificially inserted in the parental viralvector to insert the heterologous DNA.

Generally, a DNA donor vector contains the following elements: (i) aprokaryotic origin of replication, so that the vector may be amplifiedin a prokaryotic host; (ii) a gene encoding a marker which allowsselection of prokaryotic host cells that contain the vector (e.g., agene encoding antibiotic resistance); (iii) at least one DNA sequenceencoding the one or more Mtb polypeptide located adjacent to atranscriptional promoter capable of directing the expression of thesequence; and (iv) DNA sequences homologous to the region of the parentvirus genome where the foreign gene(s) will be inserted, flanking theconstruct of element (iii). Methods for constructing donor plasmids forthe introduction of multiple foreign genes into pox virus are describedin PCT Publication No. WO 91/19803, incorporated herein by reference.

Generally, DNA fragments for construction of the donor vector, includingfragments containing transcriptional promoters and fragments containingsequences homologous to the region of the parent virus genome into whichforeign DNA sequences are to be inserted, can be obtained from genomicDNA or cloned DNA fragments. The donor plasmids can be mono-, di-, ormultivalent (e.g., can contain one or more inserted foreign DNAsequences). The donor vector can contain an additional gene that encodesa marker that will allow identification of recombinant virusescontaining inserted foreign DNA. Several types of marker genes can beused to permit the identification and isolation of recombinant viruses.These include genes that encode antibiotic or chemical resistance (e.g.,see Spyropoulos et al., 1988, J. Virol. 62:1046; Falkner and Moss, 1988,J. Virol. 62:1849; Franke et al., 1985, Mol. Cell. Biol. 5:1918), aswell as genes such as the E. coli lacZ gene, that permit identificationof recombinant viral plaques by colorimetric assay (Panicali et al.,1986, Gene 47:193-199).

The DNA gene sequence to be inserted into the virus can be placed into adonor plasmid, such as an E. coli or a Salmonella plasmid construct,into which DNA homologous to a section of DNA such as that of theinsertion site of the poxvirus where the DNA is to be inserted has beeninserted. Separately the DNA gene sequence to be inserted is ligated toa promoter. The promoter-gene linkage is positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of pox DNA that isthe desired insertion region. With a parental pox viral vector, a poxpromoter is used. The resulting plasmid construct is then amplified bygrowth within E. coli bacteria and isolated. Next, the isolated plasmidcontaining the DNA gene sequence to be inserted is transfected into acell culture, for example chick embryo fibroblasts, along with theparental virus, for example poxvirus. Recombination between homologouspox DNA in the plasmid and the viral genome respectively results in arecombinant poxvirus modified by the presence of the promoter-geneconstruct in its genome, at a site that does not affect virus viability.

As noted above, the DNA sequence is inserted into a region (insertionregion) in the virus that does not affect virus viability of theresultant recombinant virus. One of skill in the art can readilyidentify such regions in a virus by, for example, randomly testingsegments of virus DNA for regions that allow recombinant formationwithout seriously affecting virus viability of the recombinant. Oneregion that can readily be used and is present in many viruses is thethymidine kinase (TK) gene. The TK gene has been found in all pox virusgenomes examined, including leporipoxvirus (Upton et al., 1986, J.Virol. 60:920); shope fibroma virus; capripoxvirus (Gershon et al.,1989, J. Gen. Virol. 70:525); Kenya sheep-1; orthopoxvirus (Weir et al.,1983, J. Virol. 46:530); vaccinia (Esposito et al., 1984, Virology135:561); monkeypox and variola virus (Hruby et al., 1983, Proc. Natl.Acad. Sci. USA 80:3411); vaccinia (Kilpatrick et al., 1985, Virology143:399); Yaba monkey tumor virus; avipoxvirus (Binns et al., 1988, J.Gen. Virol. 69:1275); fowlpox; (Boyle et al., 1987, Virology 156:355;Schnitzlein et al., 1988, J. Virol. Meth. 20:341); and entomopox (Lytvynet al., 1992, J. Gen. Virol. 73:3235-3240). In vaccinia, in addition tothe TK region, other insertion regions include, for example, the HindIIIM fragment. In fowlpox, in addition to the TK region, other insertionregions include, for example, the BamHI J fragment (Jenkins et al.,1991, AIDS Res. Hum. Retroviruses 7:991-998), the EcoRI-HindIIIfragment, EcoRV-HindIII fragment, BamHI fragment and the HindIIIfragment set forth in EPO Application No. 0 308220 A1 (see also Calvertet al., 1993, J. Virol. 67:3069-3076; Taylor et al., 1988, Vaccine6:497-503; Spehner et al., 1990, J. Virol. 64:527-533; Boursnell et al.,1990, J. Gen. Virol. 71:621-628).

In swinepox, insertion sites include the thymidine kinase gene region.In addition to the requirement that the gene be inserted into aninsertion region, successful expression of the inserted gene by themodified poxvirus requires the presence of a promoter operably linked tothe desired gene. Generally, the promoter is placed so that it islocated upstream from the gene to be expressed. Promoters are well knownin the art and can readily be selected depending on the host and thecell type to be targeted. In one example, in poxviruses, pox viralpromoters are used, such as the vaccinia 7.5K, 40K or fowlpox promoterssuch as FPV C1A. Enhancer elements can also be used in combination toincrease the level of expression. Furthermore, inducible promoters canbe utilized.

Homologous recombination between donor plasmid DNA and viral DNA in aninfected cell can result in the formation of recombinant viruses thatincorporate the desired elements. Appropriate host cells for in vivorecombination are generally eukaryotic cells that can be infected by thevirus and transfected by the plasmid vector. Examples of such cellssuitable for use with a pox virus are chick embryo fibroblasts, HuTK143(human) cells, and CV-1 and BSC-40 (both monkey kidney) cells. Infectionof cells with pox virus and transfection of these cells with plasmidvectors is accomplished by techniques standard in the art (see U.S. Pat.No. 4,603,112 and PCT Publication No. WO 89/03429).

Following in vivo recombination, recombinant viral progeny can beidentified by one of several techniques. For example, if the DNA donorvector is designed to insert foreign genes into the parent virusthymidine kinase (TK) gene, viruses containing integrated DNA will beTK⁻ and can be selected on this basis (Mackett et al., 1982, Proc. Natl.Acad. Sci. USA 79:7415). Alternatively, co-integration of a geneencoding a marker or indicator gene with the foreign gene(s) ofinterest, as described above, can be used to identify recombinantprogeny. One specific non-limiting example of an indicator gene is theE. coli lacZ gene. Recombinant viruses expressing beta-galactosidase canbe selected using a chromogenic substrate for the enzyme (Panicali etal., 1986, Gene 47:193). Once a recombinant virus has been identified, avariety of well-known methods can be used to assay the expression of theMtb sequence encoded by the inserted DNA fragment. These methods includeblack plaque assay (an in situ enzyme immunoassay performed on viralplaques), Western blot analysis, radioimmunoprecipitation (RIPA), andenzyme immunoassay (EIA).

DNA sequences encoding an Mtb polypeptide can be expressed in vitro byDNA transfer into a suitable host cell. The cell may be prokaryotic oreukaryotic. The term also includes any progeny of the subject host cell.It is understood that all progeny may not be identical to the parentalcell since there may be mutations that occur during replication. Methodsof stable transfer, meaning that the foreign DNA is continuouslymaintained in the host, are known in the art.

As noted above, a polynucleotide sequence encoding an Mtb polypeptidecan be operatively linked to expression control sequences. An expressioncontrol sequence operatively linked to a coding sequence is ligated suchthat expression of the coding sequence is achieved under conditionscompatible with the expression control sequences. The expression controlsequences include, but are not limited to, appropriate promoters,enhancers, transcription terminators, a start codon (i.e., ATG) in frontof a protein-encoding gene, splicing signal for introns, maintenance ofthe correct reading frame of that gene to permit proper translation ofmRNA, and stop codons.

Host cells can include microbial, yeast, insect and mammalian hostcells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, archea, insect, fungi(for example, yeast), mycobacterium (such as M. smegmatis), plant, andanimal cells (for example, mammalian cells, such as human). Exemplarycells of use include E. coli, Bacillus subtilis, Saccharomycescerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells,Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.Techniques for the propagation of mammalian cells in culture arewell-known (see, Jakoby and Pastan (eds), 1979, Cell Culture. Meth.Enzymol. volume 58, Academic Press, Inc., Harcourt Brace Jovanovich,N.Y.). Examples of commonly used mammalian host cell lines are VERO andHeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although othercell lines may be used, such as cells designed to provide higherexpression, desirable glycosylation patterns, or other features. Asdiscussed above, techniques for the transformation of yeast cells, suchas polyethylene glycol transformation, protoplast transformation andgene guns are also known in the art (see Gietz and Woods Meth. Enzymol.350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding an Mtbpolypeptide, and a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

In particular embodiments provided herein, one or more of the disclosedMtb polynucleotides (or fragments thereof) can be conjugated to asubstrate or solid support, such as a plate or array. In one example,the plate or array includes, consists essentially of, or consists of one(such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all)of SEQ ID NOs: 19-36 or fragments thereof. In some examples, the plateor array also includes one or more control polynucleotides. Methods forselecting an appropriate substrate and constructing a plate or array arewell known to one of skill in the art (see, e.g., U.S. Pat. Nos.5,554,501; 5,985,567; 5,981,185; and 6,013,789; and PCT Publications WO85/01051 and WO 89/10977; all incorporated herein by reference).

IV. Methods for Detecting Mtb Infection

A. Detection of T Cells

Methods for detection of a Mycobacterium infection in a subject aredisclosed herein. In several embodiments, a Mycobacterium infection canbe detected based on the presence of T cells in a biological sample,wherein the T cells specifically react with a Mtb polypeptide disclosedherein (e.g., SEQ ID NOs: 1-18).

In several embodiments, a biological sample comprising T cells isobtained from a subject of interest. Suitable biological samplesinclude, but are not limited to, blood samples, peripheral bloodmononuclear cells, sputum, saliva, cerebrospinal fluid or samples ofisolated T cells (such as CD8⁺ T cells and/or CD4⁺ T cells), lymph nodetissue, lung tissue, or other tissue sample. In one example, the sampleis incubated with a Mycobacterium polypeptide, as disclosed herein, apolynucleotide encoding the Mtb polypeptide or an APC that expresses theMtb polypeptide or a fragment thereof that binds MHC. The presence orabsence of specific activation of the T cells is detected.

The CD8⁺ T cells and/or CD4⁺ T cells which recognize the peptide in thedetection method have generally been presensitized in vivo to the Mtbpolypeptide of interest. In several embodiments, theseantigen-experienced T cells are generally present in the peripheralblood of a host which has been exposed to the antigen at a frequency of1 in 10⁶ to 1 in 10³ peripheral blood mononuclear cells (PBMCs).

In one example, the sample is isolated T cells. For example, T cells canbe isolated from a subject of interest by routine techniques (such as byFicoll/Hypaque density gradient centrifugation of peripheral bloodlymphocytes, or by fluorescence activated cell sorting). In oneembodiment the T cells used in the assay are in the form of unprocessedor diluted samples, or are freshly isolated T cells (such as in the formof freshly isolated mononuclear cells (MCs) or PBMCs which are useddirectly ex vivo, such that they are not cultured before being used inthe method. However the T cells can be cultured before use, for examplein the presence of one or more of the peptides, and generally alsoexogenous growth promoting cytokines. During culturing, the peptides aretypically presented on the surface of cells such as APCs. Pre-culturingof the T cells may lead to an increase in the sensitivity of the method.Thus the T cells can be converted into cell lines, such as short termcell lines.

In several embodiments, the T cells are incubated in vitro for two tonine days, such as about four days, at 37° C. with an Mtb polypeptide orfragment thereof that binds MHC. In several examples, the Mtbpolypeptide or fragment thereof that binds MHC is included (at aconcentration of, for example, about 5 to about 25 μg/ml, such as about5, about 10, about 15, or about 20 μg/ml). In several examples, anotheraliquot of a T cell sample can be incubated in the absence of the Mtbpolypeptide as a control.

In one embodiment, MCs are separated from the sample. The MCs includethe T cells and APCs. Thus in the method the APCs present in theseparated MCs can present the peptide to the T cells. In anotherembodiment only T cells, such as only CD8⁺ T cells, only CD4⁺ T cells,or only CD3⁺ T cells, can be purified from the sample.

The APC used in the method may be any cell which has MHC class Imolecules on its surface. It may or may not be a specialized antigenpresenting cell, such as a B cell, dendritic cell or macrophage. The APCused in the method may be from the same host as the T cell. Generally,the APC is capable of presenting the peptide to a T cell. The APC can bea freshly isolated ex vivo cell or a cultured cell such as a cell fromof a cell line.

T cells derived from the sample from the subject of interest can beplaced into an assay with all the Mtb polypeptides (or a pool of the Mtbpolypeptides, or a specific Mtb polypeptide) which it is intended totest the relevant panel or the T cells can be divided and placed intoseparate assays each of which contain one or more of the peptides. Inone embodiment, one or more of the polypeptides with an amino acidsequence set forth as SEQ ID NOs: 1-18, or a fragment of one or more ofthese polypeptides that bind MHC, is utilized. Two or more of any of theMtb peptides disclosed herein can be used for simultaneous, separate orsequential use of T cells that recognize these polypeptides. Additionalcombinations of any of the Mtb polypeptides disclosed herein can beutilized.

In one embodiment the one or more peptide(s) is (are) provided to thepresenting cell in the absence of the T cell. This cell is then providedto T cells isolated from the subject, typically after being allowed topresent the peptide on its surface.

The duration for which the peptide is contacted with the cells will varydepending on the method used for determining recognition of the peptide.Typically 10⁵ to 10⁷, such as 5×10⁵ to 10⁶ PBMCs are added to eachassay. In the case where peptide is added directly to the assay itsconcentration is typically from 10⁻¹ to 10³ μg/ml, such as about 0.5 toabout 50 μg/ml or about 1 to about 10 μg/ml. The length of time forwhich the T cells are incubated with the peptide can be from about 4 toabout 24 hours, such as from about 6 to about 16 hours, or for about 12hours.

The determination of the specific recognition of the peptide by the Tcells can be done by measuring the binding of the peptide to the Tcells. Typically T cells which bind the peptide can be sorted based onthis binding, for example using a fluorescence activated cell sorting(FACS) technique. The detection of the presence of T cells whichrecognize the peptide will be deemed to occur if the frequency of cellssorted using the peptide is above a control value.

Determination of whether the T cells recognize the peptide can also bedone by detecting a change in the state of the T cells in the presenceof the peptide or determining whether the T cells bind the peptide. Thechange in state is generally caused by antigen specific functionalactivity of the T cell after the T cell receptor binds the peptide.Generally, when binding the T cell receptor the peptide is bound to anMHC class I molecule, which may be present on the surface of a PBMC oran APC.

T cell activation can be detected by any means known to one of skill inthe art. In one example, CD8⁺ T cell activation is detected byevaluating cytolytic activity. In another example, CD8⁺ T cellactivation and/or CD4⁺ T cell activation is detected by proliferation.In several examples, a level of proliferation that is at least two foldgreater and/or a level of cytolytic activity that is at least 20%greater than in uninfected subjects indicates the presence of aMycobacterium infection in the subject of interest.

The change in state of the T cell detected may also be the start of oran increase in secretion of a substance from the T cell, such as acytokine, such as interferon (IFN)-γ, IL-2 or TNF-α. In one example, thesubstance can be detected by allowing it to bind to a specific bindingagent and then measuring the presence of the specific bindingagent/substance complex. The specific binding agent is typically anantibody, such as polyclonal or monoclonal antibodies that binds thesubstance, such as the cytokine. Antibodies to cytokines arecommercially available, or can be made using standard techniques.

Typically the specific binding agent, such as the antibody, isimmobilized on a solid support. After the cytokine is allowed to bind,the solid support can optionally be washed to remove material which isnot specifically bound to the antibody. The antibody/cytokine complexcan be detected by using a second binding agent which will bind thecomplex, such as an antibody that is labeled (either directly orindirectly) with a detectable label. Generally, the second agent bindsthe substance at a site which is different from the site which binds thefirst agent.

In several examples, the second binding agent can be detected by a thirdagent which is labeled directly or indirectly by a detectable label. Forexample the second agent may include biotin, allowing detection by athird agent which comprises a streptavidin and a label, such as anenzymatic, radioactive or fluorescent label.

In one embodiment the detection system is an ELISPOT assay, such as theassay described in PCT Publication No. WO 98/23960, incorporated hereinby reference. In one example, IFN-γ secreted from the T cell is bound bya first IFN-γ specific antibody which is immobilized on a solid support.The bound IFN-γ is then detected using a second IFN-γ specific antibodywhich is labeled with a detectable label. Exemplary labeled antibodiesare commercially available, such as from MABTECH™ (Stockholm, Sweden).An exemplary ELISPOT assay is described in the Examples section below.

The change in state of the T cell also may be an increase in the uptakeof substances by the T cell, such as the uptake of thymidine. The changein state can also be measured by an increase in the size of the T cells,or proliferation of the T cells, or a change in cell surface markers onthe T cell.

Reagents are provided herein for the detection of CD8 expressing cells(CD8⁺) that specifically bind an Mtb polypeptide as disclosed herein.These reagents are tetrameric MHC Class I/immunogenic TARP polypeptidecomplexes. These tetrameric complexes include an Mtb polypeptide, suchas a polypeptide of nine to twenty amino acids in length thatspecifically binds MHC class I.

Tetrameric MHC Class I/peptide complexes can be synthesized usingmethods well known in the art (Altmann et al., Science 274:94, 1996,which is herein incorporated by reference). In one specific non-limitingexample, purified HLA heavy chain polypeptide and β2-microglobulin (β2m)can be synthesized by means of a prokaryotic expression system. Onespecific, non-limiting example of an expression system of use is the pETsystem (R&D Systems, Minneapolis, Minn.). The heavy chain is modified bydeletion of the trans-membrane and cytosolic tail and COOH-terminaladdition of a sequence containing the biotin protein ligase (Bir-A)enzymatic biotinylation site. Heavy chain polypeptide, β2m, and peptideare then refolded. The refolded product can be isolated by any meansknown in the art, and then biotinylated by Bir-A. A tetramer is thenproduced by contacting the biotinylated product with streptavidin.

In one embodiment, the streptavidin is labeled. Suitable labels include,but are not limited to, enzymes, magnetic beads, colloidal magneticbeads, haptens, fluorochromes, metal compounds, radioactive compounds ordrugs. The enzymes that can be conjugated to streptavidin include, butare not limited to, alkaline phosphatase, peroxidase, urease andβ-galactosidase. The fluorochromes that can be conjugated to thestreptavidin include, but are not limited to, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin,allophycocyanins and Texas Red. For additional fluorochromes that can beconjugated to streptavidin, see Haugland, Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals (1992-1994). The metalcompounds that can be conjugated to the streptavidin include, but arenot limited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads. The haptens that can be conjugated to thestreptavidin include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated to streptavidin are known to the art, and include but are notlimited to technetium 99m (⁹⁹Tc), ¹²⁵I and amino acids comprising anyradionuclides, including, but not limited to, ¹⁴C, ³H and ³⁵S.Generally, streptavidin labeled with a fluorochrome is utilized in themethods disclosed herein.

In one embodiment, suspension of cells including T cells thatspecifically recognize an Mtb polypeptide is produced, and the cells arereacted with the tetramer in suspension. In one embodiment, thesereagents are used to label cells, which are then analyzed byfluorescence activated cell sorting (FACS). A machine for FACS employs aplurality of color channels, low angle and obtuse light-scatteringdetection channels, and impedance channels, among other moresophisticated levels of detection, to separate or sort cells. Any FACStechnique can be employed as long as it is not detrimental to thedetection of the desired cells. (For exemplary methods of FACS see U.S.Pat. No. 5,061,620, incorporated herein by reference).

B. Skin Tests

In another aspect, this invention provides methods for using one or moreof the polypeptides described above to diagnose Mycobacterium infection,and in particular tuberculosis, using a skin test. A “skin test” is anyassay performed directly on a patient in which a delayed-typehypersensitivity (DTH) reaction (such as induration, swelling, reddeningor dermatitis) is measured following administration into the skin, suchas the intradermal injection of one or more polypeptides describedabove. Such injection can be achieved using any suitable devicesufficient to contact the polypeptide or polypeptides with dermal cellsof the patient, such as a tuberculin syringe or 1 ml syringe. In severalexamples, the reaction is measured at least 48 hours after injection,such as between about 48 and about 72 hours after injection.

A DTH reaction is a cell-mediated immune response which is greater insubjects that have been exposed previously to the test antigen (the Mtbpolypeptide, fragment thereof that binds MHC, or fusion proteinthereof). The response can be measured visually, such as using a ruler.In several examples, a response that is greater than about 0.5 cm indiameter, such as greater than about 1.0 cm in diameter, is a positiveresponse, and is indicative of Mycobacterium infection.

The Mtb polypeptides disclosed herein can be formulated for use in askin test as pharmaceutical compositions containing a polypeptide and aphysiologically acceptable carrier. These compositions typically containone or more of the disclosed Mtb polypeptides (or a fragment thereofthat binds MHC or a fusion protein thereof) in an amount ranging fromabout 1 μg to about 100 μg, such as from about 10 μg to about 50 μg in avolume of 0.1 ml. The carrier employed in a pharmaceutical compositioncan be a saline solution with appropriate preservatives, such as phenoland/or TWEEN80™.

Generally, the polypeptide employed in a skin test is of sufficient sizesuch that it remains at the site of injection for the duration of thereaction period. In several examples, a polypeptide that is at leastnine amino acids in length is sufficient. Without being bound by theory,the polypeptide is broken down by macrophages within hours of injectionto allow presentation to T-cells. Such polypeptides can contain repeatsof one or more of the above disclosed sequences and/or other immunogenicor non-immunogenic sequences.

Thus, the determination of the recognition of the peptide by the T cellscan be measured in vivo. In several examples, the peptide isadministered to the individual and then a response which indicatesrecognition of the peptide may be measured. In one embodiment thepeptide is administered intradermally, typically in a similar manner tothe Mantoux test. The peptide can be administered epidermally. Thepeptide is typically administered by needle, such as by injection, butcan be administered by other methods such as ballistics, for example theballistics techniques which have been used to deliver nucleic acids.Published EPC Application No. EP-A-0693119 describes techniques whichcan typically be used to administer the peptide. In several examples,from 0.001 to 1000 μg, for example from 0.01 to 100 μg or 0.1 to 10 μgof peptide is administered. Alternatively an agent can be administeredwhich is capable of providing the peptides in vivo. Thus apolynucleotide capable of expressing the polypeptide can beadministered. Polypeptide is expressed from the polynucleotide in vivoand recognition of the peptide in vivo may be measured. Typically from0.001 to 1000 μg, for example from 0.01 to 100 μg or 0.1 to 10 μg ofpolynucleotide is administered.

C. Detection of Antibodies

Methods are disclosed herein wherein the polypeptides described aboveare used to diagnose Mycobacterium infection, and in particulartuberculosis. In these embodiments, methods are provided for detectingMycobacterium infection in a biological sample, using one or more of theabove polypeptides, alone or in combination. In several embodiments,multiple polypeptides are employed. The polypeptide(s) are used in anassay to determine the presence or absence of antibodies to thepolypeptide(s) in a biological sample (such as, but not limited to,whole blood, sputum, serum, plasma, saliva, or cerebrospinal fluid)relative to a control. The presence of such antibodies indicatesprevious sensitization to mycobacterial antigens which may be indicativeof Mycobacterium infection, and in particular tuberculosis.

In embodiments in which more than one polypeptide is employed, thepolypeptides can be complementary, such that one component polypeptidewill detect infection in samples where the infection would not bedetected by another component polypeptide. Complementary polypeptidesmay generally be identified by using each polypeptide individually toevaluate serum samples obtained from a series of patients known to beinfected with Mycobacterium. After determining which samples arecorrectly identified as positive with each polypeptide, combinations oftwo or more polypeptides may be formulated that are capable of detectinginfection in most, or all, of the samples tested. Complementarypolypeptides are of use to improve sensitivity of a diagnostic test.Thus, more than one of the above-described Mtb polypeptides can beincluded in an assay. Additional polypeptides from Mtb (those notdescribed herein) optionally can be included in the assay.

There are a variety of assay formats that can be used to detectantibodies in a sample (see, for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory (1988), which isincorporated herein by reference). In general, the presence or absenceof an Mtb infection in a patient may be determined by (a) contacting abiological sample obtained from a patient with one or more Mtbpolypeptides; (b) detecting in the sample the presence (or absence) ofan antibody that binds to the polypeptide(s); and (c) comparing thelevel of antibody with a control. The control can be a standard value,such as a pre-determined cut-off value. The control can be the amount ofantibodies in a subject known to be infected with Mtb, or the amount ofantibodies that specifically bind the polypeptide(s) in a subject knownnot to be infected with Mtb.

In several embodiments, the assay involves the use of a polypeptideimmobilized on a solid support. Antibodies that specifically bind thepolypeptide(s) of interest bind to the solid support. The bound antibodycan then be detected using a detection reagent that includes adetectable label. Suitable detection reagents include labeled antibodiesthat bind to the antibody/polypeptide complex. Suitable detectionreagents also include second unlabeled antibodies that bind to theantibody polypeptide complex and a third antibody that specificallybinds the second antibody. Suitable detection reagents also includeunbound polypeptide labeled with a reporter group (such as in asemi-competitive assay).

Alternatively, a competitive assay may be utilized, in which an antibodythat binds to the polypeptide of interest is labeled with a reportergroup is incubated with the sample. Following incubation, the antibodyis then allowed to bind to the immobilized antigen after incubation ofthe antigen with the sample. The extent to which components of thesample inhibit the binding of the labeled antibody to the immobilizedpolypeptide is indicative of the reactivity of the sample with theimmobilized polypeptide.

A solid support used in an assay disclosed herein can be any solidmaterial to which the antigen may be attached. For example, the solidsupport can be a test well in a microtiter plate or a nitrocellulose orother suitable membrane. Alternatively, the solid support may be a beador disc, such as glass, fiberglass, latex or a plastic material such aspolystyrene or polyvinylchloride. The support can also be a magneticparticle or a fiber optic sensor, such as those disclosed, for example,in U.S. Pat. No. 5,359,681.

The polypeptides can be bound to the solid support using a variety oftechniques. The binding of the polypeptides can be accomplished by anon-covalent association, such as adsorption, or covalent attachment,such as a direct linkage between the antigen and functional groups onthe support or a linkage through a cross-linking agent.

For binding by adsorption, binding can be achieved by contacting one ormore Mtb polypeptide(s) (generally in a buffer) with the solid supportfor a suitable amount of time. The contact time for binding is typicallybetween about 1 hour and 1 day. In general, binding is achieved bycontacting a polystyrene or polyvinylchloride solid support with anamount of the one or more Mtb polypeptide(s) ranging from about 10 ng toabout 1 μg, such as about 100 ng of antigen.

Covalent attachment of the Mtb polypeptide(s) of interest to a solidsupport can generally be achieved by reacting the support with abifunctional reagent that reacts with both the support and a functionalgroup, such as a hydroxyl or amino group, on the polypeptide. Forexample, an Mtb polypeptide can be bound to supports having anappropriate polymer coating using benzoquinone or by condensation of analdehyde group on the support with an amine and an active hydrogen onthe polypeptide (Pierce Immunotechnology Catalog and Handbook, at A12A13, 1991).

In certain embodiments, the assay is an enzyme linked immunosorbentassay (ELISA). This assay can be performed by first contacting apolypeptide antigen that has been immobilized on a solid support (suchas in the well of a microtiter plate) with the sample in a manner suchthat that antibodies present within the sample that specifically bindthe polypeptide of interest bind the immobilized polypeptide. Unboundsample is then removed and a detection reagent capable of binding to theimmobilized antibody-polypeptide complex is added. The amount ofdetection reagent that remains bound is determined using a methodappropriate for the specific detection reagent. For example, thedetection method can detect fluorescence or the presence of an enzymaticactivity.

In some embodiments, the polypeptide is immobilized on the support; anyremaining protein binding sites on the support are typically blocked.Any suitable blocking agent can be used to block the unbound proteinbinding sites, such as bovine serum albumin or TWEEN 20™. Theimmobilized polypeptide is then incubated with the sample, and theantibody is allowed to bind to the antigen. The sample can be dilutedwith a suitable diluent, for example a buffer such as phosphate-bufferedsaline (PBS) prior to incubation. In general, an appropriate contacttime (incubation time) is a period of time that is sufficient to detectthe presence of antibody in a Mycobacterium-infected sample. In onespecific, non-limiting example, the contact time is sufficient toachieve a level of binding that is at least 95% of that achieved atequilibrium between bound and unbound antibody. The time necessary toachieve equilibrium can be determined by assaying the level of bindingthat occurs over a period of time. At room temperature, an incubationtime of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% TWEEN 20™. A detectionreagent can then be added to the solid support. A detection reagent canbe any compound that binds to the immobilized antibody-polypeptidecomplex and can be detected. In several embodiments, the detectionreagent contains a binding agent (such as, for example, Protein A,Protein G, immunoglobulin, lectin or free antigen) conjugated to alabel. Labels of use include enzymes (such as horseradish peroxidase oralkaline phosphatase), substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Theconjugation of a binding agent to a label can be achieved using methodsknown in the art; conjugated binding agents are also commerciallyavailable (such as from Zymed Laboratories, San Francisco, Calif., andPierce, Rockford, Ill.).

The detection reagent is incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound antibody. An appropriate amount of time may generally bedetermined from the manufacturer's instructions or by assaying the levelof binding that occurs over a period of time. Unbound detection reagentis then removed and bound detection reagent is detected using the label.For radioactive labels, scintillation counting or autoradiographicmethods can be used for detection. Spectroscopic methods may be used todetect dyes, luminescent groups and fluorescent groups used as labels.Biotin can be detected using (strept)avidin coupled to a differentlabel, such as a radioactive label, fluorescent label or an enzymaticlabel. Enzymatic labels can be detected by the addition of substrate(generally for a specific period of time), followed by spectroscopic orother analysis of the reaction products.

To determine the presence or absence of anti-Mycobacterium antibodies inthe sample, the signal detected from the label that bound to the solidsupport is generally compared to a control. In one embodiment, thecontrol is a standard value, such as the average mean signal obtainedwhen the immobilized antigen is incubated with samples from anuninfected patient. In general, a sample generating a signal that is twoor three standard deviations above the control is considered positivefor Mycobacterium infection. In another embodiment, the control value isdetermined using a Receiver Operator Curve, according to the method ofSackett et al., Clinical Epidemiology: A Basic Science for ClinicalMedicine, Little Brown and Co., pp. 106 107 (1985). Briefly, in thisembodiment, the control value is determined from a plot of pairs of truepositive rates (sensitivity) and false positive rates (100% specificity)that correspond to each possible control value for the diagnostic testresult. The control value on the plot that encloses the largest area isthe most accurate cut-off value, and a sample generating a signal thatis higher than the cut-off value determined by this method is consideredpositive. Alternatively, the cut-off value may be shifted to minimizethe false positive rate, or to minimize the false negative rate. Ingeneral, a sample generating a signal that is higher than the cut-offvalue determined by this method is considered positive for tuberculosis.

In a related embodiment, the assay is performed in a rapid flow-throughor strip test format, wherein the antigen is immobilized on a membrane,such as, but not limited to, nitrocellulose. In a flow-through test,antibodies within the sample bind to the immobilized polypeptide as thesample passes through the membrane. A detection reagent (for example,protein A-colloidal gold) binds to the antibody-polypeptide complex asthe solution containing the detection reagent flows through themembrane. The detection of bound detection reagent can be performed asdescribed above.

In one example of the strip test format, one end of the membrane towhich the polypeptide is bound is immersed in a solution containing thesample. The sample migrates along the membrane through a regioncontaining the detection reagent and to the area of immobilizedpolypeptide. Concentration of the detection reagent at the polypeptideindicates the presence of anti-Mycobacterium antibodies in the sample.Typically, the concentration of detection reagent at that site generatesa pattern, such as a line, that can be read visually. The absence ofsuch a pattern indicates a negative result. In general, the amount ofpolypeptide immobilized on the membrane is selected to generate avisually discernible pattern when the biological sample contains a levelof antibodies that would be sufficient to generate a positive signal inan enzyme linked immunosorbant assay (ELISA). In several embodiments,the amount of polypeptide immobilized on the membrane ranges from about25 ng to about 1 μg, such as from about 50 ng to about 500 ng. Suchtests can typically be performed with a very small volume of patientserum or blood.

D. Detection of Polynucleotides

Diagnostic methods include the use of polynucleotide sequences encodingone or more of the above disclosed Mtb polypeptides. Mycobacteriuminfection can be detected by detecting the presence, absence, or levelof mRNA encoding a Mycobacterium polypeptide in a biological sample. Inseveral examples, hybridization assays are utilized, such as Northernblot or dot blot assays. In additional examples, PCR based assays areutilized.

General methods for mRNA extraction are well known in the art and aredisclosed in standard textbooks of molecular biology, including Ausubelet al., Current Protocols of Molecular Biology, John Wiley and Sons(1997). Methods for RNA extraction from paraffin embedded tissues aredisclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987),and De Andres et al., BioTechniques 18:42-44 (1995). In particular, RNAisolation can be performed using purification kit, buffer set andprotease from commercial manufacturers, such as QIAGEN®, according tothe manufacturer's instructions. For example, total RNA from cells inculture (such as those obtained from a subject) can be isolated usingQIAGEN® RNeasy mini-columns. Other commercially available RNA isolationkits include MASTERPURE™ Complete DNA and RNA Purification Kit(EPICENTRE®, Madison, Wis.), and Paraffin Block RNA Isolation Kit(Ambion, Inc., Austin, Tex.). Total RNA from tissue samples can beisolated using RNA Stat-60 (Tel-Test, Friendswood, Tex.). RNA preparedfrom a biological sample can also be isolated, for example, by cesiumchloride density gradient centrifugation.

Methods for quantitating mRNA are well known in the art. In one example,the method utilizes reverse transcriptase polymerase chain reaction(RT-PCR). Generally, the first step in gene expression profiling byRT-PCR is the reverse transcription of the RNA template into cDNA,followed by its exponential amplification in a PCR reaction. The twomost commonly used reverse transcriptases are avian myeloblastosis virusreverse transcriptase (AMV-RT) and Moloney murine leukemia virus reversetranscriptase (MMLV-RT). The reverse transcription step is typicallyprimed using specific primers, random hexamers, or oligo-dT primers,depending on the circumstances and the goal of expression profiling. Forexample, extracted RNA can be reverse-transcribed using a GeneAmp RNAPCR kit (Perkin Elmer, Calif., USA), following the manufacturer'sinstructions. The derived cDNA can then be used as a template in thesubsequent PCR reaction.

Although the PCR step can use a variety of thermostable DNA-dependentDNA polymerases, it typically employs the Taq DNA polymerase, which hasa 5′-3′ nuclease activity but lacks a 3′-5′ proofreading endonucleaseactivity. Thus, TaqMan® PCR typically utilizes the 5′-nuclease activityof Taq or Tth polymerase to hydrolyze a hybridization probe bound to itstarget amplicon, but any enzyme with equivalent 5′ nuclease activity canbe used. Two oligonucleotide primers are used to generate an amplicontypical of a PCR reaction. A third oligonucleotide, or probe, isdesigned to detect nucleotide sequence located between the two PCRprimers. The probe is non-extendible by Taq DNA polymerase enzyme, andis labeled with a reporter fluorescent dye and a quencher fluorescentdye. Any laser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the amplification reaction, the Taq DNA polymeraseenzyme cleaves the probe in a template-dependent manner. The resultantprobe fragments disassociate in solution, and signal from the releasedreporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

TAQMAN® RT-PCR can be performed using commercially available equipment,such as, for example, ABI PRISM 7700® Sequence Detection System™.(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA), orLightCycler® (Roche Applied Science, Mannheim, Germany). In oneembodiment, the 5′ nuclease procedure is run on a real-time quantitativePCR device such as the ABI PRISM 7700®. Sequence Detection System®. Thesystem includes of thermocycler, laser, charge-coupled device (CCD),camera and computer. The system amplifies samples in a 96-well format ona thermocycler. During amplification, laser-induced fluorescent signalis collected in real-time through fiber optics cables for all 96 wells,and detected at the CCD. The system includes software for running theinstrument and for analyzing the data.

In some examples, 5′-Nuclease assay data are initially expressed asC_(t), or the threshold cycle. As discussed above, fluorescence valuesare recorded during every cycle and represent the amount of productamplified to that point in the amplification reaction. The point whenthe fluorescent signal is first recorded as statistically significant isthe threshold cycle (C_(t)).

To minimize errors and the effect of sample-to-sample variation, RT-PCRcan be performed using an internal standard. The ideal internal standardis expressed at a constant level among different tissues, and isunaffected by the experimental treatment. RNAs most frequently used tonormalize patterns of gene expression are mRNAs for the housekeepinggenes glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), beta-actin, and18S ribosomal RNA.

A more recent variation of the RT-PCR technique is the real timequantitative PCR, which measures PCR product accumulation through adual-labeled fluorogenic probe (i.e., TAQMAN® probe). Real time PCR iscompatible both with quantitative competitive PCR, where internalcompetitor for each target sequence is used for normalization, and withquantitative comparative PCR using a normalization gene contained withinthe sample, or a housekeeping gene for RT-PCR (see Heid et al., GenomeRes. 6:986-994, 1996). Quantitative PCR is also described in U.S. Pat.No. 5,538,848, the disclosure of which is incorporated herein byreference. Related probes and quantitative amplification procedures aredescribed in U.S. Pat. Nos. 5,716,784 and 5,723,591, the disclosures ofwhich are incorporated herein by reference. Instruments for carrying outquantitative PCR in microtiter plates are available from PE AppliedBiosystems, 850 Lincoln Centre Drive, Foster City, Calif. 94404 underthe trademark ABI PRISM® 7700.

The steps of a representative protocol for quantitating gene expressionusing fixed, paraffin-embedded tissues as the RNA source, including mRNAisolation, purification, primer extension and amplification are given invarious published journal articles (see Godfrey et al., J. Mol. Diagn.2: 84-91, 2000; Specht et al., Am. J. Pathol. 158: 419-429, 2001).Briefly, a representative process starts with cutting about 10 μm thicksections of paraffin-embedded tissue sample. The RNA is then extracted,and protein and DNA are removed. After analysis of the RNAconcentration, RNA repair and/or amplification steps can be included, ifnecessary, and RNA is reverse transcribed using gene specific promotersfollowed by RT-PCR.

An alternative quantitative nucleic acid amplification procedure isdescribed in U.S. Pat. No. 5,219,727, which is incorporated herein byreference. In this procedure, the amount of a target sequence in asample is determined by simultaneously amplifying the target sequenceand an internal standard nucleic acid segment. The amount of amplifiedDNA from each segment is determined and compared to a standard curve todetermine the amount of the target nucleic acid segment that was presentin the sample prior to amplification.

In some embodiments of this method, the expression of a “housekeeping”gene or “internal control” can also be evaluated. These terms are meantto include any constitutively or globally expressed gene whose presenceenables an assessment of cytokine mRNA levels. Such an assessmentcomprises a determination of the overall constitutive level of genetranscription and a control for variations in RNA recovery.

E. Monitoring the Progression of an Infection and/or Effectiveness ofTherapy

In several embodiments, the diagnostic methods disclosed herein are usedfor monitoring the progression of a Mycobacterium infection. In thisembodiment, assays as described above for the diagnosis of aMycobacterium infection may be performed over time, and the change inthe level of reactive polypeptide(s) evaluated. For example, the assayscan be performed about every 12, 24, 36, 48, 60, or 72 hours for aspecified period, such as over months or weeks, and thereafter performedas needed.

In some examples, the presence of an Mtb polypeptide, or apolynucleotide encoding the polypeptide is assessed. Generally, theMycobacterium infection is progressing in those patients in whom thelevel of polypeptide (such as detected using a binding agent), the levelof polynucleotide, the level of antibodies, or the level of T cellsincreases over time. In contrast, the Mycobacterium infection is notprogressing when the level of reactive polypeptide, the level ofpolynucleotide, the level of antibodies, or the level of T cells eitherremains constant or decreases with time. In this manner, theeffectiveness of a particular therapeutic regimen can be assessed.

In one embodiment, the presence of an Mtb polypeptide or polynucleotideis assessed in a subject. The subject is administered a therapeuticprotocol. The presence of the Mtb polypeptide is then assessed. Anincrease or no change in the amount of the Mtb polypeptide (orpolynucleotide) as compared to the amount of the Mtb polypeptide priorto the administration of the therapeutic protocol indicates that thetherapeutic protocol in not effective, and the Mtb infection isprogressing. A decrease in the amount of the Mtb polypeptide (orpolynucleotide) as compared to the amount of the Mtb polypeptide (orpolynucleotide) prior to the administration of the therapeutic protocolindicates that the therapeutic protocol is effective, and that the Mtbinfection is not progressing.

In another embodiment, the presence of T cells, such as CD8⁺ T cellsand/or CD4⁺ T cells that specifically recognize an Mtb polypeptide isassessed in a subject. The subject is administered a therapeuticprotocol. The presence of the T cells that specifically recognize theMtb polypeptide is then assessed. A decrease or no change in the amountof CD8⁺ T cells and/or CD4⁺ T cells that specifically recognize the Mtbpolypeptide as compared to the amount of the CD8⁺ T cells and/or CD4⁺ Tcells, respectively, that specifically recognize the Mtb polypeptideprior to the administration of the therapeutic protocol indicates thatthe therapeutic protocol is not effective. An increase in the amount ofthe CD8⁺ T cells and/or CD4⁺ T cells that specifically recognize the Mtbpolypeptide as compared to the amount of the CD8⁺ T cells and/or CD4⁺ Tcells that specifically recognize the Mtb polypeptide prior to theadministration of the therapeutic protocol indicates that thetherapeutic protocol is effective.

It should be noted that for any of the above-described assays, toimprove sensitivity, multiple Mycobacterium markers may be assayedwithin a given sample. It will be apparent that the assays disclosedherein can be used in combination. Thus, sets of Mycobacteriumpolypeptides (or polynucleotides), and combinations of assays can be foroptimal sensitivity and specificity.

Numerous other assay protocols exist that are suitable for use with thepolypeptides of the present invention. The above descriptions areintended to be exemplary only.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Selection of Antigens

A peptide library encompassing 39,499 Mtb peptides was screened forantigens and/or epitopes that were both strongly and commonly recognizedin individuals with Mtb infection in Portland, Oreg. This peptidelibrary represents 389 genes, representing roughly 10% of the Mtbgenome. The peptides are 15 mers overlapping by 11 amino acids for eachgene product. 50 nmol of each peptide was synthesized individually andthen pooled into 777 pools of 50 peptides in a 96 well format (nineplates). Five blank wells and one well of an irrelevant peptide pool,SIV gag, were included on each of the nine plates.

CD8⁺ T cells from donors were screened against the peptide library byIFN-γ ELISPOT. The IFN-γ ELISPOT assay was performed as describedpreviously (Beckman et al., J. Immunol. 157:2795-2803, 1996). Fordetermination of ex vivo frequencies of CD8⁺ T cells responding to Mtbinfection or Mtb antigens, CD8⁺ T-cells were positively selected fromperipheral blood mononuclear cells using magnetic beads (MiltenyiBiotec, Auburn Calif.) as a source of responder T cells and tested fortheir response to autologous DC. Each plate of the genomic peptidelibrary was screened in duplicate, for a total of 18 ELISPOT plates perscreen. CD8+ T cells were prepared from cryopreserved PBMC by CD8selection using magnetic bead separations. Resulting cell populationscontained >99% CD8+ T cells. CD8+ T cells (250,000 cells/well),autologous DCs (20,000 cells/well), and IL-2 (0.5 ng/ml) were added topeptide (final 5 μg/ml, individual peptides) in the ELISPOT plates. Fivemedia control wells were included on each plate. Spots are enumeratedusing with the AID EliSpot Reader System. For each plate, the mean ofthese five wells was subtracted from each well of that plate tonormalize between plates. Each technical replicate on each plate wasthen scored. A well was scored positive if the spot forming units (SFU),less the mean of the media wells, was greater than or equal to ten andthe SFU was greater than or equal to twice the mean of the media. Twentydonors were tested, including fifteen LTBI (6 Caucasian, 4 AfricanAmerican, 5 SE Asian) and five donors with active TB.

Two criteria were used to select the peptide pools. First, peptide poolshad to be in the top 5% of a donor's response. Second, the peptide poolhad to be recognized by three or more donors. The peptide pools selectedby this method were identical independent of the order these criteriawere applied. A well was considered positive in the donor screen if onlyone technical replicate was statistically positive. However, since thereis more confidence in a well where both technical replicates arepositive, the selected wells were compared if the average spot formingunits (SFU) for wells with two positive technical replicates wasweighted by 200% to the selected wells if the average SFU was notweighted. 32 wells were selected if there was no weighting given to thetechnical replicates and 35 wells were selected if the weighting wasapplied. However, 19 wells were selected by both weighting and notweighting the average SFU and these were chosen for further analysis(Table 2).

TABLE 2 Selected antigens and epitopes for clinical validation studiesAntigen Number Rv Numbers Gene Names 1 Rv3641c (33)¹ fic 2 Rv3136(46):Rv3135 (4) PPE51:PPE50 3 Rv0383c (30):Rv0394c (20) Rv0383c:Rv0394c4 Rv1184c (20) Rv1184c 5 Rv3514 (47):Rv3532 (3) PE_PGRS57:PPE61 6 Rv3558(44):Rv3539 (6) PPE64:PPE63 7 Rv1979c (50) Rv1979c 8 Rv1980c(28):Rv1984c (22) mpt64:cfp21 9 Rv3347c (50) PPE55 10 Rv0151c (50) PE111 Rv1997 (50) ctpF 12 Rv1997 (50) ctpF 13 Rv0159c (50) PE3 14 Rv1997(50) ctpF 15 Rv2711 (37):Rv1404 (13) ideR:Rv1404 16 Rv1706c (50) PPE2317 Rv2041c (50) Rv2041c 18 Rv2041c (43):Rv2093c (7) Rv2041c:tatC 19Rv1039c (50) PPE15 ¹Number of peptides from each gene shown inparentheses

Example 2 Screening of Selected Antigens

The antigens identified in Example 1 were screened in a CD8 ELISPOTassay against latent and active TB donors from Uganda. ELISPOT plateswere read using the AID ELISPOT reader and output was exported intoexcel files. Data were imported into SAS® version 9.1 (SAS Institute,Inc., Cary, N.C.) and analyzed. A categorical variable for a positiveELISPOT was created in SAS®. For a positive response to the antigen, themean of the antigen containing wells must be greater than the backgroundwells by two standard deviations. If this was true, the background wassubtracted and this difference must then be greater than 10 spots.Similarly, a continuous ELISPOT variable was created for each antigendetailing the spot forming units remaining if the antigen met thecategorical criteria above. The results were graphed by proportion ofpositive responses stratified by active or latent TB along with thecorresponding spot forming unit (FIGS. 1A and B).

Five antigens were selected for the validation stage. Several factorswere considered in the selection, including those antigens that had asuggestion of disease specificity, as well as antigens with a broad andstrong response. These antigens included PPE50:51, PE3, CtpF, PPE15, andEsxJ. Fifty-six latent and 52 active TB individuals were studied in thevalidation phase. Twenty-one individuals (19.2%) responded to all fiveantigens at the predefined cut-off, whereas 10 individuals (9%)responded to four of the antigens. Forty individuals (36%) responded toup to three antigens and 35% did not respond to any of the five antigensselected. Although some disease specificity was noted in the screeningstage, especially as it applied to PPE50:51, this was not apparent inthe validation stage.

The magnitude of the response was studied as well. Using Poissonmodeling, individuals with latent disease had a significantly greaterspot count than those with active disease for 4 antigens (PPE50:51,cTPF, PPE15, EsXJ) however the difference was not clinically meaningful(FIG. 2).

Example 3 Additional Antigens

Additional antigens were selected using the methods described inExample 1. The additional antigens are provided in Table 3.

The additional identified antigens were screened in a CD8 ELISPOT assay(as described in Examples 1 and 2) against latent and active TB donorsfrom Uganda. The results were graphed by the corresponding spot formingunit (FIG. 3).

TABLE 3 Additional antigens and epitopes for clinical validation studiesRv_Numbers (# peptides in pool) Gene_Names Rv0284(17):Rv0288(11)Rv0284:esxH Rv0917(31) betP Rv1243c(50) PE_PGRS23 Rv3345c(100) PE_PGRS50Rv3163c(41):Rv3194c(9) Rv3163c:Rv3194c Rv0977(50) PE_PGRS16Rv0152c(40):Rv0151c(10) PE2:PE1 Rv1917c(50) PPE34Rv2040c(37):Rv2025c(13) Rv2040c:Rv2025c Rv2356c(50) PPE40 Rv3159c(50)PPE53 Rv1172c(32):Rv1195(18) PE12:PE13 Rv1348(35):Rv1343c(15)Rv1348:lprD Rv3873(50) PPE68

Example 4 Identification of Peptide-Specific T Cell Clones

Peptide-specific T cell clones were isolated from individuals with LTBIor active TB, using peptide pulsed DCs as APCs and limiting dilutioncloning methodology. Briefly, CD8+ T cells were isolated from PBMCsusing positive selection using CD8 antibody-coated magnetic beads perthe manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach,Germany). T cells were seeded at various concentrations in the presenceof a 2×10⁴-irradiated autologous peptide pulsed DC, 1×10⁵ irradiatedautologous PBMC, and rIL-2 (5 ng/ml) in cell culture media consisting of200 μl of RPMI 1640 supplemented with 10% human sera. Wells exhibitinggrowth between 10-14 days were assessed for peptide specificity usingELISPOT and peptide pulsed DCs as a source of APCs. T cells retainingpeptide specificity were further phenotyped for αβ T cell receptorexpression and CD8 expression by FACS.

Using the 15 mer Rv3136₁₃₇₋₁₅₁, T cell clones were generated to thepeptide using the methods described. Having derived an antigen-specificCD8⁺ T cell clone, the minimal epitope was determined. The minimalepitope was defined as the epitope which allowed for T cell recognitionat the lowest concentration of peptide. Each 9-mer, 10-mer, and 11-merpeptide within the 15-mer was tested over a broad range of peptideconcentrations, and by definition, the peptide eliciting a response atthe lowest peptide concentration is the minimal epitope. Peptidesincluding amino acids 139-149 of Rv3136 (SEQ ID NO: 2) allowed for Tcell recognition at the lowest concentrations (FIG. 4), with amino acids141-49 eliciting a response at the lowest concentration of all testedpeptides.

Example 5 Animal Models

In tuberculosis research, mouse and guinea pig models have been usedextensively to model various aspects of the disease.

A. Mouse Model:

Mice can be infected by a variety of routes, including intravenous,intraperitoneal and tracheal. One route is aerosolization of theinfectious organism for respiratory infection. The mice are exposed tothe aerosol in a chamber (wither whole body or nose only infection). Thedose of invention can be varied by manipulating the concentration of Mtbin the nebulizer or time of exposure. A low dose infection, such asabout 50 colony forming units (CFU) via aerosol, results in a slow andsteady increase in bacterial numbers in the lungs, generally reaching apeak in four weeks, which coincides with the peak number of T cells inthe lungs. The initial period is considered the acute stage ofinfection. Following infection, there is a dissemination of bacteria tothe mediastinal lymph nodes. T cell priming is generally detectablebetween two and three weeks. After about four weeks the bacterialnumbers stabilize, and there is a slow progressive pathologic response.This system is of use for modeling active infection. Thus, theabove-described polypeptides, or polynucleotides encoding thesepolypeptides, can be administered prior to infection. The ability of theMtb polypeptides (or polynucleotides encoding these polypeptides) toprevent infection is then assessed. Alternatively, the mice areadministered Mtb, and the ability of the Mtb polypeptide (orpolynucleotide encoding these polypeptides) to treat the Mtb infectionis monitored. The effectiveness of the Mtb polypeptides (orpolynucleotides) can be monitored by measuring the T cell response, suchas the number of CD8⁺ or CD4⁺ T cells, and/or measuring the bacterialnumbers, and/or evaluating the pathology.

Exemplary protocols are provided below (see also Repique et al., Infec.Immun. 70: 3318-3323, 2002, incorporated herein by reference for anadditional protocol).

1. Short Term Mouse Model:

C57BL/6 mice are vaccinated with a composition including one or more Mtbpolypeptide, or a polynucleotide encoding these one or more polypeptidesaccording to the appropriate protocol and then rested for 4 to 6 weeks.Immunized mice are infected with a low dose aerosol (50-100 CFU) ofvirulent M. tuberculosis and protection is evaluated by assessing thenumber of viable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of mice byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined.

BCG vaccinated mice have approximately 1 Log₁₀ protection in their lungand spleen when compared to PBS-treated mice.

B. Guinea Pig Models:

1. Short Term Guinea Pig Model

Out-bred Hartley guinea pigs are vaccinated with a composition includingone or more Mtb polypeptide, or a polynucleotide encoding these one ormore polypeptides, and then rested for 8 to 10 weeks. Immunized guineapigs are infected with a low dose aerosol (10-30 CFU) of virulent M.tuberculosis and protection is evaluated by assessing the number ofviable bacilli 30 days post challenge.

Viable counts are performed on the lung and spleen of guinea pigs byhomogenizing the organs and plating serial 10-fold dilutions on 7H11agar plates. Plates are incubated for up to 21 days and the number ofcolony forming units per organ determined. Lung and spleen segments arealso taken for histological analyses.

BCG vaccinated guinea pigs have approximately 2-3 Log₁₀ protection intheir lung and spleen when compared to PBS-treated guinea pigs. Inaddition, BCG vaccinated guinea pigs have well defined granulomas whencompared to unvaccinated animals.

2. Long Term Guinea Pig Model

The guinea pig model is similar to the mouse model, but the experimentsare open-ended survival type and can last for as long as 2 years. Guineapigs develop “classical” granulomas similar to humans with active TB,and as lung tissue necrosis progresses, they begin to lose weight anddie of TB similar to humans. The number of colony forming units in thelungs and spleen can be assessed. Histological examination can also beperformed to determine the degree of lung involvement and tissuedestruction. After low-dose aerosol exposure in the guinea pig thenumber of organisms increases progressively during the first three weeksand then plateaus into a chronic state. During the later stages ofinfection there is increased bacterial load in the lung and this isassociated with a worsening pathological condition. Without treatment,there is a concomitant rise in both CD4 and CD8 T cells in the lungs ofinfected guinea pigs.

Out-bred Hartley guinea pigs are vaccinated with the experimentalvaccine (such as a composition including one or more Mtb polypeptide, ora polynucleotide encoding these one or more polypeptides) according tothe appropriate protocol and then rested for 8 to 10 weeks. Immunizedguinea pigs are then infected with a low dose aerosol (10-30 CFU) ofvirulent M. tuberculosis. Guinea pigs are weighed weekly and monitoreddaily for signs of disease (such as increased respiration and failure tothrive). Unvaccinated guinea pigs succumb to infection from 20 to 25weeks post challenge, while BCG vaccinated guinea pigs survive for 50 to55 weeks post challenge.

At necropsy, the lung and spleen are assessed for the number of CFU andthe extent of pathology. The relative protection of the experimentalcomposition is compared to BCG vaccinated animals.

Example 6 Detection of Mtb in a Subject

This example describes exemplary methods that can be used to detectpresence Mtb in a subject. However, one skilled in the art willappreciate that methods that deviate from these specific methods canalso be used to successfully detect Mtb in a sample. In some examples,detecting Mtb diagnoses the subject as having tuberculosis or at risk ofdeveloping tuberculosis.

Clinical samples are obtained from a subject (such as a subjectsuspected of being infected with Mtb or at risk of being infected withMtb), such as a blood sample, peripheral blood mononuclear cells,sputum, saliva, or cerebrospinal fluid. In some examples, T cells areisolated from the sample by routine methods. In other examples, nucleicacids are extracted from the sample using routine methods (for exampleusing a commercial kit).

In one example, a sample including T cells from a subject are contactedwith a disclosed Mtb polypeptide (such as a polypeptide of SEQ ID NOs:1-18 or a fragment thereof), such as about 0.5 μg to 50 μg/mlpolypeptide for about 4 to 24 hours. The T cells are tested to determinewhether the polypeptide is recognized by the T cells, for example bymeasuring binding of the peptide to the T cells (for example, usingFACS, detecting T cell activation, or cytokine secretion).

In another example, a disclosed Mtb polypeptide is detected in a subjectutilizing an enzyme immunoassay such as IFA, ELISA or immunoblotting. Anexemplary ELISA method effective for the detection of Mtb antibodiescan, for example, be as follows: 1) bind a Mtb polypeptide (such as apolypeptide of SEQ ID NOs: 1-18, or a fragment thereof) to a substrate;2) contact the bound polypeptide with a fluid or tissue sample from thesubject; 3) contact the above with a secondary antibody bound to adetectable moiety which is reactive with the bound antibody (forexample, horseradish peroxidase enzyme or alkaline phosphatase enzyme);4) contact the above with the substrate for the enzyme; 5) contact theabove with a color reagent; and 6) observe/measure color change ordevelopment. In further examples, a sample from the subject is contactedwith an antibody that specifically binds one or more of the disclosedMtb polypeptides. Detection of binding of the antibody to a polypeptidein the sample (for example, utilizing a sandwich ELISA) indicates thepresence of the Mtb polypeptide in the sample from the subject.

In another example, RT-PCR is performed in a reaction including areaction mix (e.g., buffers, MgCl₂, dNTPs, and DNA polymerase), sampleRNA, and probes and primers specific for one or more Mtb polynucleotidedisclosed herein.

In some examples, detection of Mtb (such as Mtb antibodies,polypeptides, polynucleotides, or T cells that specifically react with adisclosed Mtb polypeptide) in a sample from a subject indicates that thesubject is infected with Mtb or has or is at risk of developingtuberculosis. In further examples, a therapy is selected for a subjectdiagnosed with Mtb infection, for example, antibiotic therapy.

In other examples, a disclosed Mtb polypeptide is administered to anindividual intradermally, typically in a similar manner to the Mantouxtest. The peptide is typically administered by needle, such as byinjection, but can be administered by other methods such as ballistics,for example the ballistics techniques which have been used to delivernucleic acids. In several examples, from 0.001 to 1000 μg, for examplefrom 0.01 to 100 μg or 0.1 to 10 μg of peptide is administered. Areaction (such as a delayed type hypersensitivity reaction) is measuredat least 48 hours after injection, such as between about 48 and about 72hours after injection. The response can be measured visually, such asusing a ruler. In several examples, a response that is greater thanabout 0.5 cm in diameter, such as greater than about 1.0 cm in diameter,is a positive response, and is indicative of Mycobacterium infection.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

We claim:
 1. A method for detecting a likelihood that a subject has animmune response to Mycobacterium tuberculosis, comprising contacting abiological sample from the subject comprising T cells ex vivo with oneor more Mycobacterium polypeptides, and an antigen presenting cellpresenting the one or more Mycobacterium polypeptides, wherein the aminoacid sequence of the one or more Mycobacterium polypeptides is selectedfrom the group consisting of the amino acid sequences set forth as SEQID NO: 2, SEQ ID NO: 6, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, and at least nine to twenty consecutive amino acids thereof,wherein the nine to twenty consecutive amino acids specifically bindmajor histocompatibility complex (MHC) class I; and performing anenzyme-linked immunospot assay that detects interferon-γ in order todetermine if the T cells in the biological sample specifically recognizethe amino acid sequence of the one or more Mycobacterium polypeptides,wherein a positive enzyme-linked immunospot assay determines thepresence of T cells that specifically recognize one of the Mycobacteriumpolypeptide and indicates the likelihood that the subject has an immuneresponse to Mycobacterium tuberculosis.
 2. The method of claim 1,wherein the one of the Mycobacterium polypeptides comprises or consistsof amino acids 1-15 of SEQ ID NO: 2 or amino acids 141-149 of SEQ ID NO:2.
 3. The method of claim 1, wherein the T cells are CD8+ T cells. 4.The method of claim 1, wherein the biological sample is blood, isolatedperipheral blood mononuclear cells, or isolated mononuclear cells. 5.The method of claim 1, wherein the T cells are cultured in vitro withthe one or more of the Mycobacterium polypeptides.
 6. A method ofdetecting an immune response to Mycobacterium tuberculosis in a subject,comprising; administering intradermally to the subject an effectiveamount of a Mycobacterium polypeptide, wherein the amino acid sequenceof the Mycobacterium polypeptide is selected from the group consistingof the amino acid sequences set forth as SEQ ID NO: 2, SEQ ID NO: 6, SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and at least nineto twenty consecutive amino acids thereof, wherein the nine to twentyconsecutive amino acids specifically bind major histocompatibilitycomplex (MHC) class I; after more than 48 hours following intradermaladministration, measuring induration, swelling, redness or dermatitis ofthe skin at the site of the intradermal administration, and detecting adelayed type hypersensitivity reaction of greater than 0.5 cm indiameter in the skin of the subject.
 7. The method of claim 6, whereinthe polypeptide comprises or consists of amino acids 1-15 of SEQ ID NO:2 or amino acids 141-149 of SEQ ID NO:
 2. 8. The method of claim 6,wherein the delayed type hypersensitivity reaction is greater than 1.0cm in diameter in the skin of the subject.
 9. A method for detecting alikelihood that a subject has an immune response to Mycobacteriumtuberculosis, comprising contacting a biological sample from the subjectcomprising T cells ex vivo with one or more Mycobacterium polypeptides,and an antigen presenting cell presenting the one or more Mycobacteriumpolypeptides, wherein the amino acid sequence of the one or moreMycobacterium polypeptides is selected from the group consisting of theamino acid sequences set forth as SEQ ID NO: 2, amino acids 1-15 of SEQID NO: 2, amino acids 141-149 of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and at least nine totwenty consecutive amino acids thereof, wherein the nine to twentyconsecutive amino acids specifically bind major histocompatibilitycomplex (MHC) class I; and contacting the sample with a capturemonoclonal antibody that specifically binds interferon γ, wherein thecapture monoclonal antibody is immobilized on a solid support, to form acomplex; and contacting the sample with an additional labeled antibodythat specifically binds interferon γ to detect the presence of thecomplex, wherein the presence of the labeled antibody bound to thecomplex indicates the presence of T cells that specifically recognizeone of the Mycobacterium polypeptides and indicates the likelihood thatthe subject has an immune response to Mycobacterium tuberculosis. 10.The method of claim 9, wherein the biological sample is blood, isolatedperipheral blood mononuclear cells, or isolated mononuclear cells. 11.The method of claim 9, wherein the labeled monoclonal antibody islabeled with an enzyme, a fluorescent marker, biotin or a radioactivemarker.
 12. The method of claim 11, wherein the labeled monoclonalantibody is labeled with biotin, and the method further comprisescontacting the sample with streptavidin conjugated to an enzyme, afluorescent marker or a radioactive marker.
 13. The method of claim 9,wherein the amino acid sequence of one or more of the Mycobacteriumpolypeptides is selected from the group consisting of the amino acidsequences set forth as SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 12, and SEQ ID NO: 16 and at least nine to twentyconsecutive amino acids thereof wherein the nine to twenty consecutiveamino acids specifically bind major histocompatibility complex (MHC)class I.
 14. The method of claim 9, wherein the amino acid sequence ofone or more of the Mycobacterium polypeptides is selected from the groupconsisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ IDNO: 6, and at least nine to twenty consecutive amino acids thereofwherein the nine to twenty consecutive amino acids specifically bindmajor histocompatibility complex (MHC) class I.
 15. The method of claim1, wherein the amino acid sequence of one or more of the Mycobacteriumpolypeptides is selected from the group consisting of the amino acidsequences set forth as SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 12, and SEQ ID NO: 16 and at least nine to twentyconsecutive amino acids thereof wherein the nine to twenty consecutiveamino acids specifically bind major histocompatibility complex (MHC)class I.
 16. The method of claim 1, wherein the amino acid sequence ofone or more of the Mycobacterium polypeptides is selected from the groupconsisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ IDNO: 6, and at least nine to twenty consecutive amino acids thereofwherein the nine to twenty consecutive amino acids specifically bindmajor histocompatibility complex (MHC) class I.
 17. The method of claim6, wherein the amino acid sequence of one or more of the Mycobacteriumpolypeptides is selected from the group consisting of the amino acidsequences set forth as SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 12, and SEQ ID NO: 16 and at least nine to twentyconsecutive amino acids thereof wherein the nine to twenty consecutiveamino acids specifically bind major histocompatibility complex (MHC)class I.
 18. The method of claim 6, wherein the amino acid sequence ofone or more of the Mycobacterium polypeptides is selected from the groupconsisting of the amino acid sequences set forth as SEQ ID NO: 2, SEQ IDNO: 6, and at least nine to twenty consecutive amino acids thereofwherein the nine to twenty consecutive amino acids specifically bindmajor histocompatibility complex (MHC) class I.